Isothermal methods for amplifying nucleic acid samples

ABSTRACT

The description provides two-stage methods of nucleic acid amplification and detection reactions, which are useful for rapid pathogen detection or disease diagnosis. In particular, the description provides a method comprising a first-stage slow rate amplification reaction followed by a plurality of second-stage fast rate amplification reactions that are simultaneously monitored in real-time, and wherein a rapid rate of amplification is indicative of the presence of a site of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Application Ser. No. 62,025,345 filed Jul. 16, 2014,entitled “Isothermal Methods for Amplifying Nucleic Acid Samples”, whichis incorporated herein by reference in its entirety.

BACKGROUND 1. Field of the Discovery

The description provides methods of nucleic acid amplification anddetection reactions, which are useful for rapid pathogen detection ordisease diagnosis.

2. Background Information

Nucleic acid analysis methods based on the complementarity of nucleicacid nucleotide sequences can analyze genetic traits directly.Accordingly, these methods are a very powerful means for identificationof genetic diseases, cancer, microorganisms etc. Nevertheless, thedetection of a target gene or nucleic acid present in a very smallamount in a sample is not easy, and therefore, amplification of thetarget gene or its detection signal is necessary. As such, in vitronucleic acid amplification technologies (NAATs) are an invaluable andpowerful tool for detection and analysis of small amounts of nucleicacid in many areas of research and diagnosis.

NAAT techniques allow detection and quantification of a nucleic acid ina sample with high sensitivity and specificity. NAAT techniques may beused to determine the presence of a particular template nucleic acid ina sample, as indicated by the presence of an amplification product(i.e., amplicon) following the implementation of a particular NAAT.Conversely, the absence of any amplification product indicates theabsence of template nucleic acid in the sample. Such techniques are ofgreat importance in diagnostic applications, for example, fordetermining whether a pathogen is present in a sample. Thus, NAATtechniques are useful for detection and quantification of specificnucleic acids for diagnosis of infectious and genetic diseases.

NAATs can be grouped according to the temperature requirements of theprocedure. For example, the polymerase chain reaction (PCR) is the mostpopular method as a technique of amplifying nucleic acid in vitro. Thismethod was established firmly as an excellent detection method by virtueof high sensitivity based on the effect of exponential amplification.Further, since the amplification product can be recovered as DNA, thismethod is applied widely as an important tool supporting geneticengineering techniques such as gene cloning and structuraldetermination. In the PCR method, however, temperature cycling or aspecial temperature controller is necessary for practice; theexponential progress of the amplification reaction causes a problem inquantification; and samples and reaction solutions are easilycontaminated from the outside to permit nucleic acid mixed in error tofunction as a template (See R. K. Saiki, et al. 1985. Science 230,1350-1354). Other PCR-based amplification techniques, for example,transcription-based amplification (D. Y. Kwoh, et at. 1989. Proc. Natl.Acad Sci. USA 86, 1173-1177), ligase chain reaction (LCR; D. Y. Wu, etal. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89,117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA 88, 189-193), andrestriction amplification (U.S. Pat. No. 5,102,784) similarly requiretemperature cycling.

More recently, a number of isothermal nucleic acid amplificationtechniques (iNAATs) have been developed. That is, these techniques donot rely on thermocycling to drive the amplification reaction.Isothermal amplification techniques typically utilize DNA polymeraseswith strand-displacement activity, thus eliminating the high temperaturemelt cycle that is required for PCR. This allows isothermal techniquesto be faster and more energy efficient than PCR, and also allows formore simple and thus lower cost instrumentation since rapid temperaturecycling is not required. For example, methods such as StrandDisplacement Amplification (SDA; G. T. Walker, et at. 1992. Proc. Natl.Acad. Sci. USA 89, 392-396; G. T. Walker, et al. 1992. Nuc. Acids. Res.20, 1691-1696; U.S. Pat. No. 5,648,211and EP 0 497 272, all disclosuresbeing incorporated herein by reference); self-sustained sequencereplication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci.USA 87, 1874-1878, which is incorporated herein by reference); and Qβreplicase system (P. M. Lizardi, et al. 1988. BioTechnology 6,1197-1202, which is incorporated herein by reference) are isothermalreactions (See also, Nucleic Acid Isothermal AmplificationTechnologies—A Review. Nucleosides, Nucleotides and Nucleic Acids, 2008.v27(3):224-243, which is incorporated herein by reference).

In the SDA method, a special DNA polymerase is used to synthesize acomplementary chain starting from an amplification primer complementaryto the 3′-side of a certain nucleotide sequence template, and includingone or more bumper primers upstream of the amplification primer todisplace the double-stranded chain if any at the 5′-side of the sequencetemplate. Because a double-stranded chain at the 5′-side is displaced bya newly synthesized complementary chain, this technique is called theSDA method. The temperature-changing step essential in the PCR methodcan be eliminated in the SDA method by previously insertinga-restriction enzyme recognition sequence into an annealed sequence as aprimer. That is, a nick generated by a restriction enzyme gives a 3′-OHgroup acting as the origin of synthesis of complementary chain, and thepreviously synthesized complementary chain is released as asingle-stranded chain by strand displacement synthesis and then utilizedagain as a template for subsequent synthesis of complementary chain. Inthis manner, the complicated control of temperature essential in the PCRmethod is not required in the SDA method.

In the SDA method, however, the restriction enzyme generating a nickshould be used in addition to the strand displacement-type DNApolymerase. This requirement for the additional enzyme is a major causefor higher cost. Further, because the restriction enzyme is to be usednot for cleavage of both double-stranded chains but for introduction ofa nick (that is, cleavage of only one of the chains), a dNTP derivativesuch as alpha-thio dNTP should be used as a substrate for synthesis torender the other chain resistant to digestion with the enzyme.Accordingly, the amplification product by SDA has a different structurefrom that of natural nucleic acid, and there is a limit to cleavage withrestriction enzymes or application of the amplification product to genecloning. In this respect too, there is a major cause for higher cost. Inaddition, when the SDA method is applied to an unknown sequence, thereis the possibility that the same nucleotide sequence as the restrictionenzyme recognition sequence used for introducing a nick may be presentin a region to be synthesized. In this case, it is possible that acomplete complementary chain is prevented from being synthesized.

Loop-Mediated Isothermal Amplification (LAMP) is another isothermalnucleic acid amplification technique. In LAMP, the target sequence isamplified at a constant temperature of 60 - 65 ° C. using either two orthree primer sets, and a polymerase with high strand displacementactivity in addition to a replication activity. (See Nagamine K, Hase T,Notomi T (2002). “Accelerated reaction by loop-mediated isothermalamplification using loop primers”. Mol. Cell. Probes 16 (3): 223-9; andU.S. Pat. No. 6,410,278, which is incorporated herein by reference).

LAMP was originally invented and formulated as an isothermalamplification with the strict requirement for four primers: twoloop-generating primers (FIP and BIP comprising F1, F2 and B1, B2priming sites, correspondingly) and two “Displacement primers” (F3 andB3) (FIG. 1). However, in this manifestation the LAMP technology was fartoo slow for the majority of practical applications. In order toincrease the speed of LAMP-based assays the inventors of LAMP came upwith additional “Loop primers” which, when added in conjunction with theother primers used in LAMP, resulted in significantly faster assays.Currently, the commonly used manifestation of LAMP requires a total ofsix primers: two loop-generating primers, two displacement primers andtwo “Loop primers” (L_(B) and L_(F)).

Due to the specific nature of the action of these primers, the amount ofDNA produced in LAMP is considerably higher than PCR basedamplification. The reaction can be followed in real-time either bymeasuring the turbidity or by fluorescence using intercalating dyes. Dyemolecules intercalate or directly label the DNA, and in turn can becorrelated to the number of copies initially present. Hence, LAMP canalso be quantitative. Thus, LAMP provides major advantages due to itssimplicity, ruggedness, and low cost, and has the potential to be usedas a simple screening assay in the field or at the point of care byclinicians.

Primer design for LAMP assays thus requires the selection of eightseparate regions of a target nucleic acid sequence (the FIP and BIPprimers encompass two primer binding sites each), with the BIP/FIP andLoop primers having significant restrictions on their positioningrespective to each other. “Loop primers” must be positioned strictlybetween the B2 and B1 sites and the F2 and F1 sites, respectively, andmust be orientated in one particular direction. Further, significantcare must be taken in primer design to avoid primer-dimers between thesix primers needed (especially difficult as the FIP and BIP primers aregenerally greater than 40 nucleotides long). As a consequence, LAMPprimer design is extremely challenging, especially when targeting highlypolymorphic markers and sequences containing complex secondarystructure. Also, because LAMP uses 4 (or 6) primers targeting 6 (or 8)regions within a fairly small segment of the genome, and because primerdesign is subject to numerous constraints, it is difficult to designprimer sets for LAMP “by eye”. Software is generally used to assist withLAMP primer design, although the primer design constraints mean there isless freedom to choose the target site than with PCR. In a diagnosticapplication, this must be balanced against the need to choose anappropriate target (e.g., a conserved site in a highly variable viralgenome, or a target that is specific for a particular strain ofpathogen).

LAMP has been observed to be less sensitive than PCR to inhibitors incomplex samples such as blood, likely due to use of a different DNApolymerase (typically Bst DNA polymerase rather than Taq polymerase asin PCR). LAMP is useful primarily as a diagnostic or detectiontechnique, but is not useful for cloning or myriad other molecularbiology applications enabled by PCR.

Also, multiplexing approaches for LAMP are relatively undeveloped. Thelarger number of primers per target in LAMP increases the likelihood ofprimer-primer interactions for multiplexed target sets. The product ofLAMP is a series of concatemers of the target region, giving rise to acharacteristic “ladder” or banding pattern on a gel, rather than asingle band as with PCR. Although this is not a problem when detectingsingle targets with LAMP, “traditional” (endpoint) multiplex PCRapplications wherein identity of a target is confirmed by size of a bandon a gel are not feasible with LAMP. Multiplexing in LAMP has beenachieved by choosing a target region with a restriction site, anddigesting prior to running on a gel, such that each product gives riseto a distinct size of fragment, although this approach adds complexityto the experimental design and protocol. The use of a strand-displacingDNA polymerase in LAMP also precludes the use of hydrolysis probes, e.g.TaqMan probes, which rely upon the 5′-3′ exonuclease activity of Taqpolymerase.

More recently, investigators have developed a modified LAMP techniquecalled, STEM. The LAMP-STEM system utilizes “Stem primers,” which aredirected to the stem portion of the LAMP amplicon (or “dumbbell”). Stemprimers can be used as an alternative to LAMP “Loop primers” (See FIG.2). When used in addition to loop-generating and displacement primers,Stem primers offer similar benefits in speed and sensitivity to the Loopprimers. (See Gandelman et al., Loop-Mediated Amplificaiton Acceleratedby Stem Primers. Int. J. Mol. Sci. 2011, v12:9108-9124, and US2012/0157326, which are both incorporated herein by reference). Thisbeneficial effect of Stem primers is surprising as they do not bind tothe single-stranded DNA loops, which define the very nature of the LAMPtechnology. Stem primers can be employed in either orientation, do notrequire either the B2/B1 or F2/F1 sites to be a specific distance apart,can be multiplexed, and allow the F1 and B1 sites to be positionedfurther from each other than in LAMP.

STEM primers significantly accelerate LAMP comprised of loop-generatingand displacement primers only. They can be used on their own orsynergistically with other STEM primers or even Loop primers. Additionof Stem primers into LAMP has a positive effect on both speed andsensitivity. In some cases they improve reproducibility at low copynumber. The action of Stem primers can be rationalized via the proposedmechanism of LAMP. They anneal to transiently single-stranded regions ofthe amplicon and recopy the entire binding sites for the BIP/FIPprimers. An additional unique feature is the extra strongintra-molecular self-priming when Stem primers delimit amplicon.

In general, positioning of Stem primers is less constrained than that ofLoop primers. A rather challenging primer design involving selection ofat least eight binding sites is thus simplified. Furthermore, Stemprimers impose fewer limitations on the primer design in terms of stemlength, orientation and distances between B1-B2 and F1-F2 sites. Incontradiction to the postulated LAMP mechanism that relies on theinvolvement of displacement primers Stem primers can occasionally allowdisplacement primers not to be used at all, though it is not clear whythis is so. This has a major implication for primer design, as it allowsthe ability to omit one displacement primer or even both, if necessary.

In many circumstances, such as point of care diagnostics, it isadvantageous to be able to simultaneously amplify and detect multipletargets in a single sample using a single assay. This is typically doneby combining the amplification of multiple targets in the same tubeusing different dyes attached to each different target primer set orprobe. This very common method has two significant drawbacks. One, sinceall the primers are together in one solution, there is a very highchance of them cross-reacting with each other and creating dimers andother spurious products that would interfere with the results. This isovercome by laboriously screening many combinations of primer sets tofind ones that to not cross-react.

The second major limitation of this method is that there are a limitednumber of dyes that can be separately detected when in the samesolution. Since the wavelength of the emitted light from the dye hassome bandwidth to it, each dye's emission spectrum must be adequatelyseparated from the others in order for specific and reliable detection.In practice, this limits each amplification reaction to the detection of5 or 6 different targets.

A technique that could detect a higher number of targets from the samesample without compromising sensitivity would be a huge improvement formany applications, such as a respiratory infection screening panel,where about 20 different targets are required for a thorough test.Another example is Tuberculosis, where about 20 different alleles mustbe screened in order to accurately and specifically determine thepresence of resistance to either a the two front-line drugs.

SUMMARY

The present description provides an improved nucleic acid amplificationtechnique (NAAT), which is robust, cost-effective, provides forflexibility in primer placement and design, and also demonstratesincreased rate, sensitivity, and reproducibility at low copy number. Themethods as described herein are advantageous for detecting, inreal-time, a nucleic acid region of interest, e.g., for the diagnosis ofa disease or infection.

The methods as described herein provide for a first-stage, slow-rateisothermal pre-amplification followed by multiple, discrete second-stageamplification and detection reactions performed in parallel directly onthe products from the first-stage primary amplicon amplificationproduct. At least one of the second-stage reactions includes a site orsequence-specific secondary primer (“site-specific primer”), wherein ifthe template comprises a complementary site for the site-specificprimer, the site-specific primer amplification reaction proceeds at afaster rate relative to both the first-stage reaction and second-stagereaction in which no secondary primer is added. In certain embodiments,at least one of the second-stage reactions includes a secondary primerhaving a base pair mismatch (“mismatch primer”), e.g., 3′ mismatchnucleotide (“3′ mismatch primer”), wherein the site-specific primeramplification reaction proceeds at a faster rate relative to thefirst-stage reaction, second-stage reactions in which no secondaryprimer is added, and the second-stage mismatch primer reaction.

For example, a first-stage region-specific pre-amplification step isdone then the reaction is split into multiple reaction chambers, wherein each chamber a different set of secondary primers, e.g., loop or stemprimers, are introduced that are site-specific sensitive by virtue of,e.g., a 3′ end match/mismatch, or annealing temperature difference, sothat a much higher speed exponential reaction only occurs if thesecondary primers are correctly matched to the sample sequence. Thesignificant difference in amplification rate can be detected byreal-time fluorescent measurement of, e.g., an intercalating dye, andthus multiple site-specific reactions within the same region of interestcan be individually identified with a single assay.

Thus, in one aspect, the description provides two-stage nucleic acidamplification reaction comprising: providing a target nucleic acidtemplate and at least one primer that anneals to the target nucleic acidtemplate near a region of interest to be amplified; performing afirst-stage nucleic acid amplification (or “pre-amp”) reaction toamplify the region of interest (amplicon). In certain embodiments, aforward and reverse primer are provided and used to synthesize andamplify the region of interest or amplicon of interest.

Subsequently, the pre-amp reaction product (“amplified region ofinterest”) is utilized in one or more second-stage amplificationreactions wherein at leaset one of the reactions includes asite-specific secondary primer, such that rapid amplification occursonly if a complementary sequence for the site-specific primer (i.e., asite-specific region of interest) exists in the amplified region ofinterest from stage-one (i.e., the nucleic acid region of interest fromstage-one is positive for the site of interest). As described herein,the appearance of amplification products in each second-stage reactioncan be detected and compared simultaneously and in real-time, wherein afast rate of amplification relative to the first-stage reaction, asecond-stage reaction in which no secondary primer is added, and asecond-stage mismatch primer reaction is indicative of the presence ofthe site-specific region of interest.

In certain embodiments, the site-specific primer comprises a nucleotidemismatch at a site other than the 3′ terminus.

In certain embodiments, multiple second-stage nucleic acid amplificationreactions are performed in parallel. In a preferred embodiment, thesite-specific primer reaction is performed and monitored in parallelwith a separate reaction in which similar site-specific primers are usedbut that comprise a mismatch in its nucleic acid sequence.

In another aspect, a method is described comprising performing multiple,separate or discrete second-stage amplification reactions utilizing thefirst-stage amplification product or primary amplicon in which the rateof the second-stage amplification reaction is detected and compared asbetween at least one reaction having a site-specific primercomplementary to a site or sequence of interest on the primary amplicontemplate, and a reaction having a secondary primer that anneals to thesame site but comprises a base-pair mismatch (herein, “a mismatchprimer”), wherein a faster reaction rate relative to the other isindicative of the presence or absence of the specific site or region ofinterest. In still additional embodiments, the rate of the second-stageamplification reaction is detected and compared as between at least onereaction having a site-specific primer complementary to a site orsequence of interest on the primary amplicon template, a reaction havinga mismatch primer, and a reaction that has no secondary primer, whereina faster reaction rate relative to the other reactions is indicative ofthe presence or absence of the specific site or region of interest.

In certain embodiments, the mismatch primer is a mismatch Stem primer.In any of the embodiments described herein, the mismatch primercomprises at least one mismatched nucleotide (i.e., a nucleotide that isnot complementary to the template). In certain embodiments, themismatched nucleotide is at the 3′ end of the mismatch primer (“3′mismatch primer).

In certain embodiments, the site-specific primer and the mismatch primercomprise at their 3′ ends a nucleotide that is specific or complementaryfor a multi-allelic or polymorphic site such that, depending on whichallele or polymorphism the nucleic acid template comprises, thesite-specific primer could comprise a 3′ end nucleotide mismatch, andthe mismatch primer could contain the 3′ end complementary nucleotide.In other words, in certain embodiments, the method comprises performinga plurality of second-stage amplification reactions, wherein eachreaction comprises a site-specific primer. However, as described herein,only those amplification reactions comprising a primer that iscomplementary at its 3′ end to the site of interest will proceed at anincreased rate relative to the first-stage reaction, and relative to thesecond-stage reaction comprising a 3′ mismatch.

In certain embodiments, the method comprises performing a plurality ofsecond-stage amplification reactions, wherein each respective reactioncomprises a site-specific primer. In certain embodiments, thesite-specific primer is specific for a different multi-allelic orpolymporphic site. In such a configuration, the methods provide for amultiplexing method of determining the presence or absence of multiplepolymorphisms simultaneously.

In certain embodiments, the method includes performing at least oneadditional second-stage amplification reaction in parallel in which noadditional primers are added, and monitoring and comparing in real-timethe rate of the second-stage amplification reaction as between thereaction comprising the site-specific primer, the reaction comprisingthe mismatch primer, and the reaction comprising no primer, wherein anenhanced reaction rate relative to the others is indicative of thepresence of the specific site or region of interest.

In certain embodiments, the method comprises performing a plurality ofseparate or discrete second-stage amplification reactions approximatelysimultaneously. In certain additional embodiments, the discretereactions comprise the same or different site-specific primers. Incertain embodiments, the discrete reactions comprise a plurality orcombination of site-specific primers (i.e., “multiplexing reaction”).

Although several of the aspects and embodiments refer to a two-stagedamplification scheme, the invention is not so limited. Indeed, themethods described herein are predicated upon the surprising discoverythat the presence of a specific site or sequence of interest in a targetnucleic acid region can be detected by the enhanced rate ofamplification with a site-specific or sequence-specific (i.e.,complementary) primer relative to the slower rate of an amplificationreaction with mismatch primer that, but-for the mismatched base, annealsto the same target region. As such, any number of additionalamplification steps can be performed, e.g., 3, 4, 5, 6, 7, 8, 9, etc.,and therefore, any number of sites, regions or sequences of interest canbe detected. Similarly, for each of the respective site-specific primerreactions to be performed, a parallel mismatch primer reaction can beperformed and the rate of amplification of the two reactions monitored.

In any of the aspects or embodiments described herein, the first-stageamplification reaction can comprise site-specific primers such that thefirst-stage pre-amplification reaction is selective for a particularsite of interest, and only if that site or region exists in the samplewill rapid amplification of the template proceed at the second or laterstage amplification reaction.

In any of the aspects or embodiments described herein, the second-stage(or later stage) amplification reaction can comprise multiplesite-specific primers (i.e., “multiplex reaction”). By usingdifferentially labeled site-specific oligonucleotides differentamplification products can be detected and compared (i.e., multiplexed)within the same reaction.

In another aspect, the description provides a method for detecting andcomparing nucleic acid amplification comprising providing a firstreaction chamber; performing a first-stage pre-amplification reaction asdescribed herein in the first reaction chamber; introducing an amount orvolume of the first-stage pre-amp reaction directly into a plurality ofsecond reaction chambers, wherein at least one second reaction chambercomprises a site-specific primer, and at least one second reactionchamber comprises a mismatched primer; and performing in each secondreaction chamber a second-stage amplification reaction as descriedherein; and detecting and comparing the rate of amplification of eachreaction simultaneously, wherein a faster rate of amplification in thesite-specific primer reaction versus the mismatch primer reaction isindicative of the presence of the site of interest.

In any of the embodiments described herein, the amplification reactionsof stage-one and stage-two are performed sequentially in the samereaction container or chamber. In certain embodiments, the amplificationreactions are performed sequentially but in different reactioncontainers or chambers. In a preferred embodiment, the second-stagereaction is performed in parallel in a plurality of containers orchambers, and detected and compared.

In any of the embodiments described herein, the target nucleic acidtemplate is comprised in a suitable buffer and introduced into acontainer comprising loop-forming primers. In still additionalembodiments, the mixture from the first-stage amplification reaction isintroduced into a container comprising site-specific or mismatchprimers.

In additional embodiments, the first-stage amplification reaction isperformed in a container or chamber comprising a channel that is influid communication with one or more additional containers or chamberscomprising primers for performing the second-stage amplificationreaction (i.e., site-specific primer, site-specific Stem primer;mismatch primer, mismatch Stem primer, etc). In still additionalembodiments, the first-stage reaction chamber comprises a channel thatis in one-way fluid communication with one or more additional containersor chambers comprising primers for performing the second-stageamplification reaction.

In any of the aspects or embodiments described herein, the first-stageamplification reaction is an isothermal nucleic acid amplificationreaction. In certain embodiments, the second-stage amplificationreaction is an isothermal nucleic acid amplification reaction. Inadditional embodiments, both the first-stage amplification reaction, andthe second-stage amplification reaction are isothermal nucleic acidamplification reactions. In certain embodiments, the first-stage and/orsecond-stage amplification reaction is a LAMP-based isothermalamplification reaction. In a preferred embodiment, the first-stageamplification is a LAMP-based isothermal amplification reaction and thesecond-stage is a LAMP-STEM-based isothermal amplification reaction.

In any of the aspects or embodiments described herein, the mismatchprimer comprises a 3′ end terminal nucleotide mismatch. In certainembodiments, a site-specific primer as described herein comprises on the3′ terminal end a nucleotide complementary to a nucleotide of interestin the target nucleic acid template.

In another aspect the description provides a two-stage method ofdiagnosing a genetic disease, SNP, viral or microbial infection whereinsaid method comprises providing a nucleic acid sample from subject to betested, performing the two-stage amplification reaction as describedherein, wherein the site-specific primer is specific for a region ornucleotide(s) of interest associated with at least one of a geneticdisease, mutation, SNP, virus, or bacteria, and wherein an increasedrate of amplification with the site-specific primer versus a mismatchprimer is indicative of a subject having an allele for the geneticdisease, mutation, SNP, virus or microbe. In certain embodiments, themicrobe is a bacterium.

In certain embodiments, the method of diagnosing a disease or pathogenicinfection comprises the step of treating the patient testing positivefor the nucleotide of interest with an appropriate therapeutic, e.g.,antibiotic, drug, etc.

In any of the aspects or embodiments described herein, the methodfurther comprises a step of detecting the formation of an amplicon. Incertain embodiments, the amplification reaction mixture comprises afluorescently labeled dNTPs or a fluorescent DNA intercalating dye. Incertain embodiments, formation of an amplicon is detected in real-timeby measuring an increase in fluorescence.

In any of the aspects or embodiments described herein, the nucleic acidtemplate comprises a plurality of primer binding regions, wherein theprimers serve as origins of synthesis and define a polynucleotide region(i.e., “an amplicon”) to be amplified using a polymerase.

In any of the embodiments described herein, the method further comprisesthe addition of at least one Displacement or bumper primer in theamplification reaction.

In any of the embodiments described herein, the target nucleic acidtemplate comprises genomic DNA, cDNA or RNA or a segment therefrom, froma virus, plant, microbe, or multicellular organism, e.g., a mammal, suchas a human. In certain embodiments, the genomic DNA is from a pathogenicvirus or microbe, e.g., bacteria or archae. In certain embodiments, thetarget nucleic acid template is from tubercle bacillus (MTB or TB). Incertain additional embodimetns, the target nucleic acid template is fromthe rpoB gene from MTB. In still further embodiments, the target nucleicacid template is rpoB13.5 F6.

In any of the embodiments described herein, the method further comprisesthe addition of at least one loop primer which is complementary to aregion in the first loop or the second loop of the amplicon.

In any of the embodiments described herein, the first-amplificationreaction is preceeded by a heating step in which the target nucleic acidtemplate and primers are heated to approximately 95° C. for from about 1minute to about 15 mintues. In certain embodiments, the target nucleicacid template and primers are heated to approximately 95° C. for fromabout 5 minutes to about 10 minutes.

In any of the embodiments described herein, the amplification reactionis performed at a temperature of from about 50° C. to about 75° C. Incertain embodiments, the amplification reaction is performed at atemperature of from about 55° C. to about 65° C.

In any of the embodiments described herein, the site-specific primer(e.g., site-specific Stem primer) is in the forward direction. Incertain embodiments, the site-specific primer is in the reversedirection. In still additional embodiments, both forward and reversesite-specific primers are included in the reaction.

In any of the embodiments described herein, 3′ SNP specific nucleotideis comprised within at least one of a loop-forming primer, adisplacement primer, or a loop primer.

In any of the embodiments described herein, the primers or nucleotidereagents comprise a chemical modification.

In any of the embodiments described herein, the first-stage andsecond-stage amplification reactions are performed sequentially in thesame reaction container or chamber. In certain embodiments, thefirst-stage and second-stage amplification reactions (and any subsequentamplification reactions) are performed sequentially but in differentreaction chambers.

In any of the embodiments described herein, the primers can comprise anendonuclease restriction site, or recognition element for a nickingenzyme.

In any of the embodimetns described herein, the amplification reactioncan comprise a hairpin primer comprising a first and a second segment,wherein the first segment is substantially complementary to a primerbinding region on a template and the second segment comprises a sequencethat is substantially complementary to another region in the primer;

In any of the embodiments described herein, the amplification reactioncan comprise a loop-providing primer, comprising a hairpin primer inwhich the inverted repeats are separated by a linker region.

In any of the embodiments described herein, the amplification reactioncan comprise a chimeric primer.

The preceding general areas of utility are given by way of example onlyand are not intended to be limiting on the scope of the presentdisclosure and appended claims. Additional objects and advantagesassociated with the compositions, methods, and processes of the presentinvention will be appreciated by one of ordinary skill in the art inlight of the instant claims, description, and examples. For example, thevarious aspects and embodiments of the invention may be utilized innumerous combinations, all of which are expressly contemplated by thepresent description. These additional advantages objects and embodimentsare expressly included within the scope of the present invention. Thepublications and other materials used herein to illuminate thebackground of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated byreference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating an embodiment of the invention and are not to be construedas limiting the invention. Further objects, features and advantages ofthe invention will become apparent from the following detaileddescription taken in conjunction with the accompanying figures showingillustrative embodiments of the invention, in which:

FIG. 1. Illustration of general principle for loop-mediated isothermalamplification (LAMP). In an exemplary method as described herein a firststage isothermal amplification reaction based on the LAMP method isperformed, which includes FIP and BIP “loop-forming primers” (LFPs)(comprising F1c and F2, B1c and B2, priming sites, respectively), and“displacement primers” (F3 and B3). The LAMP reaction producesconcatamers of the “dumbbell” structure (c). As described herein, theinitial LAMP reaction proceeds at a relatively slow rate.

FIG. 2. Illustration of general principal for an exemplary modifiedLAMP-STEM technique as described herein. As described herein, aLAMP-STEM amplication reaction is performed as the second-stage. Thereaction includes at least one “site-specific Stem primer,” which iscomplementary to a location or sequence of interest in the amplicon fromthe stage-one amplification reaction. The site-speific Stem primerreaction is performed in parallel with separate amplification reactioncomprising a Stem primer having a mismatch, e.g., a mismatch at its 3′end (“mismatch Stem primer”); and the two reactions are simultaneouslymonitored and compared, wherein an incrase in the amplification rate ofthe site-specific Stem primer reaction relative to the mismatch Stemprimer reaction is indicative of the presence of the site of interest inthe amplicon (or concatmers thereof), and wherein it is indicative ofthe absence of the site of interest when the rate of the site-specificStem primer reaction is the same or slower than the mismatch Stem primerreaction.

FIG. 3. Illustration of general principal for another exemplary modifiedLAMP-STEM technique as described herein. The technique is similar tothat depicted in FIG. 2 but futher includes “Loop primers” in additionto the SNP-Stem primers. See WO0028082, incorporated herein byreference.

FIG. 4. Illustrates an exemplary system configured for performing thetwo-stage amplification method as described herein. In the example, thefirst-stage reaction is performed in a central chamber, which is incommunication, e.g., fluid communication, with one or more additionalchambers. In a preferred embodiment, the first-stage reaction mixture isdistributed evenly to the one or more than one additional chambers wherethe second-stage amplification reaction takes place.

FIG. 5. Real-time theoretical signal for 10² and 10⁶ starting copies and10× second-stage amplification efficiency versus first-stage reactionefficiency. The upper panel shows the two exponential reactions rates ona log scale, and the lower panel shows them on a linear scale. Thevertical axis represents the number of copies of amplicon present; bothinput material and amplified material. This axis also represents thesignal that can be generated and detected using an intercalating dye.The dashed horizontal line represents the limit of detection of aninstrument that is measuring the fluorescence signal that can begenerated by said dye. The horizontal axisof both graphs represent timein miniutes. The 8-minute time point is the point at which thesecond-stage primers are added to the reaction. This is where theaccelerated reaction starts if the second-stage primer(s) are matched toa site within the primary amplicon that was generated by the firstreaction, which is represented by the lines with increased slopes in thelog chart. The continuation of the lower slope lines on the log chartindicates the signal that would be present if the secondary primers donot match the site of interest within the amplicon.

FIG. 6. Illlustrates the effect on rate of amplification due tomismatched bases in Stem primers. (a) The left panel exemplifies a Stemprimer comprising a 3′ end mismatch representing a primer that is notspecific for a SNP of interest. The 3′ mismatch induces a delay in theamplification reaction versus the matched or SNP-specific Stem primer(“SNP-Stem primer”). The increase in rate of amplification isexemplified in the right panel, wherein the dotted line represents theearliest time (t) to the point of maximum slope of fluorescence(t_(max)1), and ΔCt1 represents the lag in time to t_(max) between theSNP-Stem primer and the 3′ end mismatched Stem primer. (b) The rightpanel exemplifies another embodiment as described herein, wherein theStem primers (SNP-Stem primer and 3′ end mismatched primer) containanother base pair mismatch in their sequence. The second mismatch shiftsto the right (i.e., reduces the rate) of amplification (ΔCt2) and delaysthe time to t_(max)2.

FIG. 7. Processed fluorescent traces showing matched Stem primer (i.e.,SNP-Stem primer) LAMP reactions (n=4), and mismatched Stem primer LAMPreactions (n=4) using the Bio-Rad CFX realtime PCR instrument.

FIG. 8. Illustrates the enhanced rate of amplification observedaccording to an exemplary method as described herein. (a) Provides rawtraces showing matched Stem primer (i.e., SNP-STEM) reactions (lighttraces) and mismatched Stem primer reactions (dark traces). (b)Quantification cycle (Cq; See Hellemans et al., 2007) is measured fromthe fitted raw traces as the point of maximum slope. The Cq for matchedprimers is plotted (light dots) along with mismatched Stem primers (darkdots) and no Stem primer LAMP (“dumbbell”) reactions (open circles).

DETAILED DESCRIPTION

The following is a detailed description provided to aid those skilled inthe art in practicing the present invention. Those of ordinary skill inthe art may make modifications and variations in the embodimentsdescribed herein without departing from the spirit or scope of thepresent disclosure. All publications, patent applications, patents,figures and other references mentioned herein are expressly incorporatedby reference in their entirety.

The present description provides an improved nucleic acid amplificationtechnique (NAAT), and in particular iNAAT, which is robust,cost-effective, provides for flexibility in primer placement and design,and also demonstrates increased rate, sensitivity, and reproducibilityat low copy number, in particular, as compared to, e.g., SDA or LAMP.

As described above, it is advantageous to have a platform that separatesamplification samples into multiple separate reactions, so there is nointerference of dyes or primers, so that more tests can be reliablyperformed per sample. In order to do this without diluting the sampleand thus reducing sensitivity or inducing non-uniformity between themultiple reaction chambers, the nucleic acids in the sample should beamplified somewhat before being distributed to the final reaction areas.This will ensure that all the downstream reaction chambers have anadequate amount of starting DNA (or RNA) to reliably deliver ameaningful result.

A known way to do this is to use an initial PCR reaction that amplifiesa product that contains the regions of interest but is somewhat larger,and then distributes that product to separate downstream reactions thatonly amplify each small region of interest (or SNP). This is known asnested PCR, since each target sequence or SNP is “nested” within alarger amplified region. If this method is used, a dilution step istypically required so that the pre-amplification primers do notcontaminate the final reaction and detection step. A dilution step ishighly undesirable in an automated instrument because it requires anadditional fluid source and additional manipulations of the samplefluid. It is far simpler and therefore more favorable to be able to usethe fluid containing the pre-amplified sample directly in the finalamplification and detection reaction.

Another way to do this is to use Irma instead of Thymine in thepre-amplification primers, and then destroy the primers in a subsequentstep using an enzyme (that destroys the Uracil), and then using theresulting pre-amp'd but primer-free solution for the final amplificationand detection step. This method has the advantage of not requiring theintroduction of a dilutent, but requires an additional enzyme, which maybe provided dry and immobilized (lyophilized), which may be simpler thana dilution step: it is further desirable to use a method that does notrequire temperature cycling (isothermal). This is because isothermalamplification reactions tend to be faster than temperature cyclingreactions (such as PCR), and because isothermal requirements result in alower cost and lower power instrument.

Another known method for pre-amplifying a sample is to use whole genomeamplification, whereby all of the genetic material in the sample. isamplified equally. This is accomplished using a library of short (6 or 7base) primers that represent nearly all possible sequences, and can thuscover all genomes. Nearly all nucleic acid samples will be contaminatedwith nucleic acids from organisms other than the ones of interest, andsometimes there is a factor of over 10⁸ excess of this unwanted geneticmaterial than of the genetic material of interest.

Therefore, for this method, the vast majority of the amplificationresources required for this method of pre-amplification will be consumedby the amplification of unwanted, contaminating DNAs. Also, within thenucleic acids of interest, only a tiny portion (0.01% or less typically)of the genome is tested for in the final amplification reaction, so thewhole genome amplification method is further miss-directed atnon-informative genomic areas by a factor of over 10¹². In addition,whole genome amplification methods take a relatively long time period(hours), and require special, highly processive strand-displacingenzymes, at high concentrations, which add cost to the assay. If toomuch material is pre-amplified using this method, which could happen ina highly contaminated sample, then a positive signal could result in thefinal detection area, even if no DNA of interest was present. A dilutionstep would be required to prevent this false positive signal, which addscost and complexity to the fluid handling system of an instrumentemploying this technology.

A method that has neither of the above additional assay requirements andtheir associated drawbacks is preferred.

The methods as described herein provide for a low-rate isothermalpre-amplification followed by high rate site-specific amplification anddetection step. For instance, an exemplary method as described hereinincludes a first-stage slow rate isothermal reaction followedimmediately by a separate or second-stage fast rate exponentialisothermal amplification reaction that proceeds at an enhanced raterelative to the first reaction.

In another example, a regionspecific pre-amplification step is doneusing the LAMP system without the presence of loop primers or stemprimers, thus resulting in a very slow exponential reaction speed. Oncean amplification of approximately 100-100,000× is achieved (in apredetermined time frame), the reaction is split into multiple reactionchambers, where in each chamber a different set of loop or stem primersare introduced that are site-specific sensitive by virtue of, e.g., a 3′end match/mismatch, or annealing temperature difference, so that themuch higher speed exponential reaction only occurs if the loop or stemprimers are correctly matched to the sample sequence. The significantdifference in amplification rate can be detected by real-timefluorescent measurement of, e.g., an intercalating dye, and thusmultiple site-specific reactions within the same region of interest canbe individually identified with a single assay.

Thus, in one aspect, the description provides two-stage nucleic acidamplification reaction comprising: providing a target nucleic acidtemplate and at least one primer that anneals to the target nucleic acidtemplate near a region of interest to be amplified; performing afirst-stage nucleic acid amplification (or “pre-amp”) reaction toamplify the region of interest (amplicon). In certain embodiments, aforward and reverse primer are provided and used to synthesize andamplify the region of interest or amplicon of interest.

Subsequently, the pre-amp reaction product (“primary amplicon” or“amplified region of interest”) is utilized in one or more second-stageamplification reactions wherein at leaset one of the reactions includesa site or sequence-specific secondary primer (“site-specific primer”),such that rapid amplification occurs only if a complementary sequencefor the site-specific primer (i.e., a site-specific region of interest)exists in the primary amplicon or amplified region of interest fromstage-one. In other words, the nucleic acid region of interest fromstage-one is positive for the site of interest. As described herein, theappearance of amplification products in each second-stage reaction canbe detected and compared simultaneously and in real-time, wherein a fastrate of amplification relative to the first-stage reaction, asecond-stage reaction in which no secondary primer is added, and asecond-stage mismatch primer reaction is indicative of the presence ofthe site-specific region of interest.

Significantly, because in certain embodimetns, the region beingamplified is the same in both the first-stage and second-stagereactions, it is advantageous to introduce the second-stageamplification reaction primers well before there is a measurable productfrom the first -stage amplification reaction, otherwise the second-stageamplification product cannot be detected.

The methods as described herein provide for a first-stage, slow-rateisothermal pre-amplification followed by multiple, discrete second-stageamplification and detection reactions performed in parallel directly onthe products from the first-stage amplification. At least one of thesecond-stage reactions includes a site-specific secondary primer,wherein if the primary amplicon template comprises a complementary sitefor the site-specific primer, the site-specific primer amplificationreaction proceeds at a faster rate relative to both the first-stagereaction and second-stage reaction in which no secondary primer isadded. In certain embodiments, at least one of the second-stagereactions includes a secondary primer having a mismatch nucleotide(“mismatch primer”), e.g., a 3′ mismatch (“3′ mismatch primer”), whereinthe site-specific primer amplification reaction proceeds at a fasterrate relative to the first-stage reaction, second-stage reactions inwhich no secondary primer is added, and the second-stage mismatch primerreaction.

For example, the description provides a method wherein a first-stageregion-specific pre-amplification step is performed, and then thereaction is split into multiple reaction chambers, where in at least onechamber a secondary primer, e.g., loop or stem primer, is introducedthat is site-specific sensitive by virtue of, e.g., a 3′ endmatch/mismatch, or annealing temperature difference, so that a muchhigher speed exponential reaction only occurs if the secondary primer iscorrectly matched to the sample sequence. The significant difference inamplification rate can be detected by real-time fluorescent measurementof, e.g., an intercalating dye, and thus multiple site-specificreactions within the same region of interest can be individuallyidentified with a single assay.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription is for describing particular embodiments only and is notintended to be limiting of the invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms in which case each carbon atomnumber falling within the range is provided), between the upper andlower limit of that range and any other stated or intervening value inthat stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

The following terms are used to describe the present invention. Ininstances where a term is not specifically defined herein, that term isgiven an art-recognized meaning by those of ordinary skill applying thatterm in context to its use in describing the present invention.

The articles “a” and “an” as used herein and in the appended claims areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article unless the context clearlyindicates otherwise. By way of example, “an element” means one elementor more than one element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.”

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of’ and “consistingessentially of’ shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from anyone or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anonlimiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, in certain methods described hereinthat include more than one step or act, the order of the steps or actsof the method is not necessarily limited to the order in which the stepsor acts of the method are recited unless the context indicatesotherwise.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein and includestautomers, regioisomers, geometric isomers, and where applicable,stereoisomers, including optical isomers (enantiomers) and othersteroisomers (diastereomers) thereof, as well as pharmaceuticallyacceptable salts and derivatives (including prodrug forms) thereof whereapplicable, in context. Within its use in context, the term compoundgenerally refers to a single compound, but also may include othercompounds such as stereoisomers, regioisomers and/or optical isomers(including racemic mixtures) as well as specific enantiomers orenantiomerically enriched mixtures of disclosed compounds. The term alsorefers, in context to prodrug forms of compounds which have beenmodified to facilitate the administration and delivery of compounds to asite of activity. It is noted that in describing the present compounds,numerous substituents and variables associated with same, among others,are described. It is understood by those of ordinary skill thatmolecules which are described herein are stable compounds as generallydescribed hereunder. When the bond is shown, both a double bond andsingle bond are represented within the context of the compound shown.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human or a domesticated animal, to whomtreatment, including prophylactic treatment, with the compositionsaccording to the present invention is provided. For treatment of thoseinfections, conditions or disease states which are specific for aspecific animal such as a human patient, the term patient refers to thatspecific animal, including a domesticated animal such as a dog or cat ora farm animal such as a horse, cow, sheep, etc. In general, in thepresent invention, the term patient refers to a human patient unlessotherwise stated or implied from the context of the use of the term.

The term “effective” is used to describe an amount of a compound,composition or component which, when used within the context of itsintended use, effects an intended result. The term effective subsumesall other effective amount or effective concentration terms, which areotherwise described or used in the present application.

The terms “nucleic acid,” “polynucleotides,” and “oligonucleotides”refers to biopolymers of nucleotides and, unless the context indicatesotherwise, includes modified and unmodified nucleotides, and both DNAand RNA. For example, in certain embodiments, the nucleic acid is apeptide nucleic acid (PNA). Typically, the methods as described hereinare performed using DNA as the nucleic acid template for amplification.However, nucleic acid whose nucleotide is replaced by an artificialderivative or modified nucleic acid from natural DNA or RNA is alsoincluded in the nucleic acid of the present invention insofar as itfunctions as a template for synthesis of complementary chain. Thenucleic acid of the present invention is generally contained in abiological sample. The biological sample includes animal, plant ormicrobial tissues, cells, cultures and excretions, or extractstherefrom. In certain aspects, the biological sample includesintracellular parasitic genomic DNA or RNA such as virus or mycoplasma.The nucleic acid may be derived from nucleic acid contained in saidbiological sample. For example, genomic DNA, or cDNA synthesized frommRNA, or nucleic acid amplified on the basis of nucleic acid derivedfrom the biological sample, are preferably used in the describedmethods.

The term “primer” or “oligonucleotide primer” can refer to a shortpolynucleotide that satisfies the requirements that it must be able toform complementary base pairing sufficient to anneal to a desirednucleic acid template, and give an —OH group serving as the origin ofsynthesis of complementary chain at the 3′-terminal. Accordingly, itsbackbone is not necessarily limited to the one via phosphodiesterlinkages. For example, it may be composed of a phosphothioate derivativehaving S in place of O as a backbone or a peptide nucleic acid based onpeptide linkages. The bases may be those capable of complementary basepairing. In the nature, there are 5 bases, that is, A, C, T, G and U,but the base can be an analogue such as bromodeoxyuridine. Theoligonucleotides as used herein can function not only as the origin ofsynthesis but also as a template for synthesis of complementary chain.The term polynucleotide includes oligonucleotides, which have arelatively short chain length. Signficantly, a primer need not be fullycomplementary in order to anneal to a binding site on a polynucleicacid.

In certain embodiments, the primer is complementary to the binding sitesequence. In other embodiments, the primer comprises one or moremismatched nucleotides (i.e., bases that are not complementary to thebinding site). In still other embodiments, the primer can comprise asegment that does not anneal to the polynucleic acid or that iscomplementary to the inverse strand of the polynucleic acid to which theprimer anneals. In certain embodiments, a primer is 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 ,24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length. In apreferred embodiment, the primer comprises from 2 to 100 nucleotides.

Oligonucleotides can be used to hybridize with genomic DNA or cDNAsequences in the same or different organisms. Those of skill in the artwill appreciate that various degrees of stringency of hybridization canbe employed in the assay. The degree of complementarity (sequenceidentity) required for detectable binding will vary in accordance withthe stringency of the hybridization medium and/or wash medium. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. Minorsequence variations in the probes and primers can be compensated for byreducing the stringency of the hybridization.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) or hybridize with another nucleic acid sequence byeither traditional Watson-Crick or other non-traditional types. As usedherein “hybridization,” refers to the binding, duplexing, or hybridizingof a molecule only to a particular nucleotide sequence under low,medium, or highly stringent conditions, including when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. See e.g.Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, N.Y., 1993

If a nucleotide at a certain position of a polynucleotide is capable offorming a Watson-Crick pairing with a nucleotide at the same position inan anti-parallel DNA or RNA strand, then the polynucleotide and the DNAor RNA molecule are complementary to each other at that position. Thepolynucleotide and the DNA or RNA molecule are “substantiallycomplementary” to each other when a sufficient number of correspondingpositions in each molecule are occupied by nucleotides that canhybridize or anneal with each other in order to effect the desiredprocess. A complementary sequence is a sequence capable of annealingunder stringent conditions to provide a 3′-terminal serving as theorigin of synthesis of complementary chain.

“Forward primer binding site” and “reverse primer binding site” refersto the regions on the template DNA and/or the amplicon to which theforward and reverse primers bind. The primers act to delimit the regionof the original template polynucleotide which is exponentially amplifiedduring amplification. In some embodiments, additional primers may bindto the region 5′ of the forward primer and/or reverse primers. Wheresuch additional primers are used, the forward primer binding site and/orthe reverse primer binding site may encompass the binding regions ofthese additional primers as well as the binding regions of the primersthemselves. For example, in some embodiments, the method may use one ormore additional primers which bind to a region that lies 5′ of theforward and/or reverse primer binding region. Such a method wasdisclosed, for example, in WO0028082 which discloses the use of“displacement primers” or “outer primers”.

The term “template” used in the present invention means nucleic acidserving as a template for synthesizing a complementary chain in anucleic acid amplification technique. A complementary chain having anucleotide sequence complementary to the template has a meaning as achain corresponding to the template, but the relationship between thetwo is merely relative. That is, according to the methods describedherein a chain synthesized as the complementary chain can function againas a template. That is, the complementary chain can become a template.In certain embodiments, the template is derived from a biologicalsample, e.g., plant, animal, virus, micro-organism, bacteria, fungus,etc. In certain embodiments, the animal is a mammal, e.g., a humanpatient.

“Patient sample” refers to any sample taken from a patient and caninclude blood, stool, swabs, sputum, Broncho Alveolar Lavage Fluid,tissue samples, urine or spinal fluids. Other suitable patient samplesand methods of extracting them are well known to those of skill in theart. A “patient” or “subject” from whom the sample is taken may be ahuman or a non-human animal. When a sample is not specifically referredto as a patient sample, the term also comprises samples taken from othersources. Examples include swabs from surfaces, water samples (forexample waste water, marine water, lake water, drinking water), foodsamples, cosmetic products, pharmaceutical products, fermentationproducts, cell and micro-organism cultures and other samples in whichthe detection of a micro-organism is desirable.

In the present invention, the terms “synthesis” and “amplification” ofnucleic acid are used. The synthesis of nucleic acid in the presentinvention means the elongation or extension of nucleic acid from anoligonucleotide serving as the origin of synthesis. If not only thissynthesis but also the formation of other nucleic acid and theelongation or extension reaction of this formed nucleic acid occurcontinuously, a series of these reactions is comprehensively calledamplification.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as determined by the match between strings of such sequences.“Identity” and “similarity” can be readily calculated by known methods,including, but not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., Siam J. Applied Math., 48:1073 (1988). In addition, values forpercentage identity can be obtained from amino acid and nucleotidesequence alignments generated using the default settings for the AlignXcomponent of Vector NTI Suite 8.0 (Informax, Frederick, Md.).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215:403-410 (1990)). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLMNIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). The well-known Smith Waterman algorithm may also be used todetermine identity.

A “single-nucleotide polymorphism” (SNP) is a DNA sequence variationoccurring commonly within a population (e.g. 1%) in which a singlenucleotide—A, T, C or G—in the genome (or other shared sequence) differsbetween members of a biological species or paired chromosomes. Forexample, two sequenced DNA fragments from different individuals, AAGCCTAto AAGCTTA, contain a difference in a single nucleotide (i.e., there aretwo alleles). Almost all common SNPs have only two alleles. SNPs canoccur in coding, non-coding or intergenic regions of DNA . The genomicdistribution of SNPs is not homogenous; SNPs occur in non-coding regionsmore frequently than in coding regions or, in general, where naturalselection is acting and ‘fixing’ the allele (eliminating other variants)of the SNP that constitutes the most favorable genetic adaptation. Otherfactors, like genetic recombination and mutation rate, can alsodetermine SNP densi. SNPs within a coding sequence do not necessarilychange the amino acid sequence of the protein that is produced, due todegeneracy of the genetic code.

SNPs in the coding region are of two types, synonymous and nonsynonymousSNPs. Synonymous SNPs do not affect the protein sequence whilenonsynonymous SNPs change the amino acid sequence of protein. Thenonsynonymous SNPs are of two types: missense and nonsense. SNPs thatare not in protein-coding regions may still affect gene splicing,transcription factor binding, messenger RNA degradation, or the sequenceof non-coding RNA. Gene expression affected by this type of SNP isreferred to as an eSNP (expression SNP) and may be upstream ordownstream from the gene.

As there are for genes, bioinformatics databases exist for SNPs. dbSNPis a SNP database from the National Center for Biotechnology Information(NCBI). Kaviar is a compendium of SNPs from multiple data sourcesincluding dbSNP. SNPedia is a wiki-style database supporting personalgenome annotation, interpretation and analysis. The OMIM databasedescribes the association between polymorphisms and diseases (e.g.,gives diseases in text form), the Human Gene Mutation Database providesgene mutations causing or associated with human inherited diseases andfunctional SNPs, and GWAS Central allows users to visually interrogatethe actual summary-level association data in one or more genome-wideassociation studies. The International SNP Map working group mapped thesequence flanking each SNP by alignment to the genomic sequence oflarge-insert clones in Genebank. Another database is the InternationalHapMap Project, where researchers are identifying Tag SNP to be able todetermine the collection of haplotypes present in each subject.

The polynucleic acid produced by the amplification technology employedis generically referred to as an “amplicon” or “amplification product.”The nature of amplicon produced varies significantly depending on theNAAT being practised. For example, NAATs such as PCR may produceamplicon which is substantially of identical size and sequence. OtherNAATs produce amplicon of very varied size wherein the amplicon iscomposed of different numbers of repeated sequences such that theamplicon is a collection of concatamers of different length. Therepeating sequence from such concatamers will reflect the sequence ofthe polynucleic acid which is the subject of the assay being performed.

In the present specification, the simple expression “5′-side” or“3′-side” refers to that of a nucleic acid chain serving as a template,wherein the 5′ end generally includes a phosphate group and a 3′ endgenerally includes a free —OH group.

The term “disease state or condition” is used to describe any diseasestate or condition, in particular, those relating to geneticabnormalities or due to the presence of a pathogenic organism such as avirus, bacteria, archae, protozoa or multicellular organism.

Methods

As described herein, it has been surprisingly and unexpectedlydiscovered that performing a two-stage nucleic acid amplificationreaction using primers that provide for differential reaction ratesdepending on the presence or absence of specific sequences of interestallows for the rapid, real-time detection of nucleic acid sequences ofinterest, which provides advantages for point-of-care diagnosis.

In one aspect, the description provides two-stage nucleic acidamplification reaction comprising: providing a target nucleic acidtemplate and at least one primer that anneals to the target nucleic acidtemplate near a region of interest to be amplified; performing afirst-stage nucleic acid amplification (or “pre-amp”) reaction toamplify the region of interest (amplicon). In certain embodiments, aforward and reverse primer are provided and used to synthesize andamplify the region of interest or amplicon of interest.

Subsequently, the first-stage pre-amp reaction product (“primaryamplicon” or “amplified region of interest”) is utilized in one or morediscrete second-stage nucleic acid amplification reactions, wherein inat least one second-stage reactiona site-specific primer is included,such that rapid or accelerated amplification occurs only if acomplementary sequence for the site-specific primer (i.e., site-specificregion of interest) exists in the primary amplicon. Stated differently,if the rate of second-stage amplification reaction is acceleratedrelative to the stage-one reaction, and any second-stage reactionslacking an additional primer, and/or second-stage reactions having amismatch primer, the nucleic acid region of interest from stage-one ispositive for the site of interest.

Thus, according to certain methods described herein, the rate of thesecond-stage reaction is selectively enhanced relative to the first bythe presence of specific primer binding sequences that are fullycomplementary or at least complementary to the 3′ end of thesecond-stage primer (herein, the “site-specific primer”). In a certainembodiments, the relative amplification reaction rates are as follows:second-stage site-specific primer amplification>>first-stageamplification reaction. In still additional embodiments, the relativeamplification reaction rates are as follows: second-stage site-specificprimer amplification>>first-stage reaction second-stage mismatch primeramplification. As used herein, the term “mismatch primer” generallyrefers to a primer having a 3′ end nucleotide that is not complementaryto the primer binding site on the template.

As indicated above, the differential relative amplification reactionrates between the first-stage, and second-stage, as well as between thesecond-stage site-specific primer versus second-stage mismatch primerare important for being able to selectively detect for the presence(i.e., amplification) of the site of interest. For example, FIG. 5demonstrates that the first-stage pre-amplification reaction proceedsrelatively slowly to the threshold signal level (i.e., the Cq islonger). However, in the presence of a complementary primer, thesecond-stage amplification reaction proceeds exponentially (i.e., the Cqfor the complementary primer is faster than that of both the first-stagereaction, and second-stage mismatch primer reaction).

In certain embodiments, the second-stage amplification rate isexponential. In certain embodiments of the methods described herein, thesecond-stage site-specific primer reaction rate is at least about 5, 10,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,500, 1000, 10000, 100000 or more times faster than the second-stagemismatch primer reaction. In certain embodiments of the methodsdescribed herein, the second-stage site-specific primer reaction rate isabout 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 500, 1000, 10000, 100000 or more times faster than thesecond-stage mismatch primer reaction.

In certain embodiments of the methods described herein, the resulting Cq(time to positive detection of the reaction) for the second-stagesite-specific primer reaction is at least about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,500%, 1000%, 10000% or more faster than the second-stage mismatch primerreaction. In certain embodiments of the methods described herein, theresulting Cq (time to positive detection of the reaction) for thesecond-stage site-specific primer reaction is about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 500%, 1000%, 10000% or more faster than the second-stage mismatchprimer reaction. In certain embodiments of the methods described herein,the resulting Cq (time to positive detection of the reaction) for thesecond-stage site-specific primer reaction is at least about 25% fasterthan the second-stage mismatch primer reaction. In certain embodimentsof the methods described herein, the resulting Cq (time to positivedetection of the reaction) for the second-stage site-specific primerreaction is from about 10% to about 1000%, from about 20% to about 500%,or from about 25% to about 100% faster than the second-stage mismatchprimer reaction. However, the important factor is that the differentialin time be far enough above the variability of the system to confidentlydistinguish which second-stage reaction is matched (i.e., thesecond-stage primer is complementary to the primer binding site on thetemplate, in particular at the 3′ end).

As described below, in certain embodiments the first-stage amplificationreaction is a relatively slow LAMP-based amplification. In additionalembodiments, the second-stage amplification reaction is a relativelyrapid LAMP-STEM-based amplification, wherein the reaction rate isenhanced by the binding of site-specific Stem primers to the site ofinterest. As such, the methods as described herein provide for robust,rapid, efficient, and highly specific amplification of desired geneticsequences. The amplification of the desired amplicons can be monitoredor detected by any known method, including, e.g., intercalating dyes,labeled reagents, etc.

In certain embodiments, the site-specific primer is a site-specific Stemprimer. In certain embodiments, the mismatch primer is a mismatch Stemprimer. In any of the embodiments described herein, the mismatch primercomprises at least one mismatched nucleotide (i.e., a nucleotide that isnot complementary to the template). In certain embodiments, themismatched nucleotide is at the 3′ end of the mismatch primer (“3′mismatch primer”).

In certain embodiments, the site-specific primer and the mismatch primercomprise at their 3′ ends, respectively, a nucleotide that is specificor complementary to one form of a multi-allelic or polymorphic site suchthat, depending on which allele or polymorphism the nucleic acidtemplate comprises, the site-specific primer could comprise a 3′ endnucleotide mismatch, and the mismatch primer could contain the 3′ endcomplementary nucleotide. In other words, in certain embodiments, themethod comprises performing a plurality of second-stage amplificationreactions, wherein each reaction comprises a primer that is specific fora particular allele. However, as described herein, only thoseamplification reactions comprising a secondary primer that iscomplementary at its 3′ end to the site of interest will proceed at anincreased rate relative to the first-stage reaction, and relative to thesecond-stage reaction comprising a 3′ mismatch.

In certain embodiments, the method comprises performing a plurality ofsecond-stage amplification reactions, wherein each respective reactioncomprises a site-specific or allele-specific primer. In certainembodiments, each site-specific primer in each of the respectivesecond-stage amplification reactions is specific for a different allele,mutation or polymporphism. In such a configuration, the methods providefor a multiplexing method for determining the presence or absence ofmultiple polymorphisms simultaneously. The methods as described are,therefore, particularly advantageous for differentiating betweenmulti-allelic genes, genotypes, clades, forms, groups, subgroups,classes, species or strains. For example, certain pathogenic bacteriaare closely related to non-pathogenic strains or species. As such, in anexemplary aspect, the current description provides methods comprisignmultiple second-stage reactions, wherein each respective reactioncomprises a primer (or primer pair) that is specific for a site orallele of interest that allows for the simultaneous determination of,e.g., species and strain of a microbe.

For example, in one embodiment, the description provides a two-stagenucleic acid amplification and real-time detection method comprising

-   -   a. providing a target nucleic acid template and at least one        primer that anneals to the target nucleic acid template near a        region of interest to be amplified;    -   b. performing a first-stage nucleic acid amplification reaction        to amplify the region of interest, thereby forming aprimary        amplicon;    -   c. dividing (b) into at least tworeactions, including in at        least one of the reactions a secondary site-specific primer that        is complementary to a site-specific primer binding region (i.e.,        a site-specific region of interest) in the primary amplicon and        defines a site of interest, and including at least onereaction        in which no secondary primer is included;    -   d. performing a second nucleic acid amplification reaction        thereby amplifying the region of interest; and    -   e. detecting and comparing in real-time the amplification rate        of the site-specific primer reaction and the mismatch primer        reaction, wherein an enhanced rate of amplification in the        site-specific primer reaction relative to the mismatch primer        reaction is indicative of the presence of the site of interest.        In certain embodiments, a forward and reverse primer are        provided and used to synthesize and amplify the region of        interest or amplicon of interest.

In any of the aspects or embodiments described herein, the mismatchprimer comprises a 3′ end terminal nucleotide mismatch. In any of theaspects or embodiments described herein, a site-specific primer cancomprise on the 3′ terminal end a nucleotide that is complementary to anucleotide of interest in the target nucleic acid template. In certainembodiments, the site-specific primer comprises a nucleotide mismatch ata site other than the 3′ terminus.

In certain embodiments, multiple second-stage nucleic acid amplificationreactions are performed in parallel. In a preferred embodiment, thesite-specific primer reaction is performed and monitored in parallelwith a separate reaction in which similar site-specific primers are usedbut that comprise a mismatch in its nucleic acid sequence. In otherwords, in one reaction the site-specific primer is fully complementaryto a site of interest on the target nucleic acid, and in anotherreaction, the primer anneals to the same site but comprises a mismatch,e.g., a 3′ end mismatch. The inventors have discovered that a mismatchin the site-specific primer slows the amplification reaction rate enoughsuch that by direct, real-time detection or montoring and comparing ofboth amplifications reactions in parallel it is possible to identify thepresence or absence of sequence variances that exist in the nucleic acidsample. Stated differently, the rate of the second amplificationreaction is selectively enhanced or suppressed based on the presence ofa sequence that is complementary to the site-specific primer. Withoutbeing bound by any particular theory, it is believed that the rate ofthe second-stage amplification is enhanced by the use of thesite-specific primer because it serves as another origin of extension.

In another aspect, a method is described comprising performing multiple,separate or discrete second-stage amplification reactions utilizing thefirst-stage amplification product in which the rate of the second-stageamplification reaction is compared as between at least one reactionhaving a site-specific primer complementary to a region or sequence ofinterest on the template, and a reaction having a primer that anneals tothe same site but comprises a base-pair mismatch (herein, “a mismatchprimer”), wherein a faster reaction rate relative to the other isindicative of the presence or absence of the specific site or region ofinterest.

In certain embodiments, the method includes performing at least oneadditional second-stage amplification reaction in parallel with thesite-specific and mismatch amplification reactions, wherein noadditional primers are added, and monitoring and comparing in real-timethe rate of the second-stage amplification reaction comprising thesite-specific primer, the reaction comprising the mismatch primer, andthe reaction comprising no primer, wherein an enhanced reaction raterelative to the others is indicative of the presence of the specificsite or region of interest.

The target template used in the present invention may be any polynucleicacid that comprises suitable primer binding regions that allow foramplification of a polynucleic acid of interest. The skilled person willunderstand that the forward and reverse primer binding sites need to bepositioned in such a manner on the target template that the forwardprimer binding region and the reverse primer binding region arepositioned 5′ of the sequence which is to be amplified on the sense andantisense strand, respectively.

The target template may be single or double stranded. Where the targettemplate is a single stranded polynucleic acid, the skilled person willunderstand that the target template will initially comprise only oneprimer binding region. However, the binding of the first primer willresult in synthesis of a complementary strand which will then containthe second primer binding region.

The target template may be derived from an RNA molecule, in which casethe RNA needs to be transcribed into DNA before practising the method ofthe invention. Suitable reagents for transcribing the RNA are well knownin the art and include, but are not limited to, reverse transcriptase.

The methods as described herein can be practiced with any NAAT. Forexample, known methods of DNA or RNA amplification include, but are notlimited to, polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis, et at; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor, et al; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson, et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No.5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, etal; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 toBiswas; U.S. Pat. No. 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al, with the tradename NASBA), the entire contents of whichreferences are incorporated herein by reference. (See, e.g., Ausubel,supra; or Sambrook, supra.).

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

In certain aspects, the NAAT that is utilized in the methods describedherein are isothermal nucleic acid amplification techniques. Someisothermal amplification techniques are dependent on transcription aspart of the amplification process, for example Nucleic Acid SequenceBased Amplification (NASBA; U.S. Pat. No. 5,409,818) and TranscriptionMediated Amplification (TMA; U.S. Pat. No. 5,399,491) while others aredependent on the action of a Helicase or Recombinase for exampleHelicase Dependent Amplification (HDA; WO2004027025) and Recombinasepolymerase amplification (RPA; WO03072805) respectively, others stillare dependent on the strand displacement activity of certain DNApolymerases, for example Strand Displacement Amplification (SDA; U.S.Pat. No. 5,455,166), Loop-mediated Isothermal Amplification (LAMP;WO0028082, WO0134790, WO0224902), Chimera Displacement Reaction (RDC;WO9794126), Rolling Circle Amplification (RCA; Lizardi, P. M. et al.Nature Genetics, (1998) 19.225-231), Isothermal Chimeric Amplificationof Nucleic Acids (ICAN; WO0216639), SMart Amplification Process (SMAP;WO2005063977), Linear Isothermal Multimerization Amplification (LIMA;Isothermal amplification and multimerization of DNA by Bst DNApolymerase, Hafner G. J., Yang I. C., Wolter L. C., Stafford M. R.,Giffard P. M, BioTechniques, 2001, vol. 30, no4, pp. 852-867) alsomethods as described in U.S. Pat. No. 6,743,605 (herein referred to as‘Template Re-priming Amplification’ or TRA) and WO9601327 (hereinreferred to as ‘Self Extending Amplification’ or SEA).

A characteristic of these NAATs is that they provide for both copying ofa polynucleic acid via the action of a primer or set of primers and forre-copying of said copy by a reverse primer or set of primers. Thisenables the generation of copies of the original polynucleic acid at anexponential rate. With reference to NAATs in general it is helpful todifferentiate between the physical piece of nucleic acid being detectedby the method, from the first copy made of this original nucleic acid,from the first copy of the copy made from this original nucleic acid,from further copies of this copy of a copy. For the sake of clarity, thenucleic acid whose provenance is from the sample being analysed itselfwill be referred to as the “target nucleic acid template.” Withreferenct to the two-stage method described herein, the first-stageprimer-dependent amplification reaction is relatively slow as comparedto the second-stage reaction.

As would be understood by the skilled artisan, the primer-generatedamplicon gives rise to further generations of amplicons through repeatedamplification reactions of the target nucleic acid template as well aspriming of the amplicons themselves. It is possible for amplicons to becomprised of combinations with the target template.

The amplicon may be of very variable length as the target template canbe copied from the first priming site beyond the region of nucleic aciddelineated by the primers employed in a particular NAAT. In general, akey feature of the NAAT will be to provide a method by which theamplicon can be made available to another primer employed by themethodology so as to generate (over repeated amplification reactions)amplicons that will be of a discrete length delineated by the primersused. Again, a key feature of the NAAT is to provide a method by whichthe amplicons are available for further priming by a reverse primer inorder to generate further copies. For some NAATs, the later generationamplicons may be substantially different from the first generationamplicon, in particular, the formed amplicon may be a concatamer of thefirst generation amplicon.

Methods which produce amplicons in the form of concatamers directly fromlinear target templates include LAMP (including, LAMP-STEM), TRA, SEAand SMAP (the latter is a hybrid of LAMP and SEA). In each case theconcatamers arise from processes involving the first generationamplicon. Thus, it is preferred that synthesis of the polynucleic acidis performed using a NAAT selected from the group consisting of LAMP,TRA, SEA and SMAP. In each case therefore, the invention is associatedwith a NAAT which provides one or more primers with the capability ofproducing a concatamer directly from a linear target template.

The methods as described are preferably perfomed with a NAAT thatresults in the formation of template concatamers. The term “concatamer”as used herein refers to a polynucleic acid having substantially similarnucleotide sequences linked alternately in a single-stranded chain.These arrayed sequences may be simple repeats of each other, invertedrepeats or combinations thereof. Further, the process is understood tobe repeated by the next generation amplicons such that longer and longerconcatamers can be formed.

NAATs which are suitable for the generation of concatamers are wellknown in the art and generally include “isothermal” amplificationtechniques. This means that the amplification of the polynucleic aciddoes not require a change in the incubation temperature, contrary toknown thermocycling techniques, such as polymerase chain reaction. Forexample, isothermal amplification methods include, e.g., SDA, LAMP,LAMP-STEM as described above, can be used in conjunction with themethods described herein.

A common feature of LAMP, TRA, SMAP and SEA is therefore that of firstgeneration amplicon dependent priming, i.e. where the first generationamplicon acts as a primer itself, whether by an intra-molecular event orinter-molecular event, leading to next generation amplicon that islarger in size than the first generation amplicon and which isconcatameric in nature. In fact, it is a characteristic of these NAATsthat longer and longer amplicon is generated from shorter amplicon suchthat the number of binding sites for stem primers increasesexponentially during the amplification process and hence the ability forstem primers to accelerate amplification. Appreciation of the mechanismof action of the primers generating the concatamers in these NAATs ishelpful in understanding how stem primers have their effect.Furthermore, the skilled person aware of the mechanisms which lead togeneration of a concatamer will readily be able to identify othersuitable NAATs which can be used in the methods of the presentinvention.

In certain embodiments described herein, the first-stage amplificationreaction is an isothermal nucleic acid amplification reaction. Incertain embodiments, the second-stage amplification reaction is anisothermal nucleic acid amplification reaction. In additionalembodiments, both the first-stage amplification reaction, and thesecond-stage amplification reaction are isothermal nucleic acidamplification reactions. In certain embodiments, the first-stage and/orsecond-stage amplification reaction is a LAMP-based isothermalamplification reaction. In a preferred embodiment, the first-stageamplification is a LAMP-based isothermal amplification reaction and thesecond-stage is a LAMP-STEM-based isothermal amplification reaction,wherein the site-specific primer accelerates the amplification rate(relative to the first amplification reaction), and allows for earlierdetection of amplification products relative to the 3′ mismatchedprimer.

In certain embodiments, In addition to the forward and reverse primerbinding regions, the target template needs to comprise a stem regionthat needs to have a sufficient length to allow binding of the one ormore stem primers (e.g., site-specific Stem primer or mismatch Stemprimer), as described herein. Thus it is preferred that the stem regionhas a length of at least 5 nucleotides, at least 10 nucleotides, atleast 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides,at least 50 nucleotides, at least 100 nucleotides at least 200nucleotides, at least 300 nucleotides or at least 500 nucleotides.

WO0028082 describes the use of loop-forming primers (LFPs), where a LFPis understood to comprise a first and second segment, wherein the firstsegment is substantially complementary to a primer binding region on thetemplate and the second segment comprises a sequence that issubstantially complementary to a region in the amplicon generated fromthe first segment of the first primer such that the second segment isable to form a loop, and mentions that the NAAT uses two “outer primers”in addition to the LFPs. These primers are characterised in that the“first outer primer” binds 3′ to the “F2” site in the template (i.e. thefirst outer primer binds the “F3” site, FIG. 1) and the “second outerprimer” binds 3′ to the binding region of the second LFP, the “R2c” site(i.e. the second outer primer binds the “R3c” site, FIG. 1). Thus, theseprimers do not bind in the stem-region of the amplicon, which lies 5′ ofthe primer binding sites of the LFPs.

A primer employed in LAMP generates single stranded loops in theamplicon and is referred to herein as “loop forming primer.”Loop-forming primer, as used herein, refers to primers which comprise afirst and a second segment, wherein the first segment is substantiallycomplementary to the primer binding region on the template and thesecond segment comprises a sequence that is substantially complementaryto a region in the amplicon generated from the first segment of thefirst primer such that the second segment is able to form a loop. Thegeneral structure of loop-forming primers is shown in FIG. 1. The first(and next) generation amplicon resulting from the priming of the targettemplate by a loop-forming primr contains a loop of single strandedpolynucleotide flanked by double-stranded polynucleotide formed fromWatson-Crick base-pairing of the inverted repeat sequence. Thesingle-stranded loop of polynucleotide is understood to be available forbinding by a further primer employed by the NAAT in question butspecifically not by a stem primer.

The primer oligonucleotides the present description can be prepared byany method known in the art, including by direct chemical synthesis(see, e.g., Ausubel, et al., supra). Chemical synthesis generallyproduces a single-stranded oligonucleotide, which can be converted intodouble-stranded DNA by hybridization with a complementary sequence or bypolymerization with a DNA polymerase using the single strand as atemplate. One of skill in the art will recognize that while chemicalsynthesis of DNA can be limited to sequences of about 100 or more bases,longer sequences can be obtained by the ligation of shorter sequences.

Nucleic acid extension is facilitated by a DNA polymerase, for example,a thermostable strand-displacing (SD) DNA polymerase. Because of thethermostable SD polymerase, there is no need for thermocycling.Displacement or bumper primers (F3 and B3) can be included to facilitatedisplacement of the FIP and BIP primers, and allow more efficientextension of the complementary DNA strand.

The region between the forward and reverse primer binding regionsrepresents a region which is guaranteed to form part of the amplicon butdoes not itself conventionally provide for any primer binding sites.This region is referred to herein as the “stem region” of the amplicon.Primers which bind to the stem region are referred to herein as “stemprimers.” Stem primers can be defined as primers which bind to the stemregion (FIG. 2). They may further be defined as primers that bind theregion 3′ of the forward primer binding region on the forward strand and3′ of the reverse primer binding site on the reverse strand. It isunderstood that the primer binding sites and the binding sites of thestem primers do not significantly overlap. It is preferred that theprimer binding sites and the binding sites of the stem primers do notoverlap at all.

“Significantly” in the context of overlapping primer binding regionsmeans that the primer binding sites overlap by less than 10 nucleotides,less than 9 nucleotides, less than 8 nucleotides, less than 7nucleotides, less than 6 nucleotides, less than 5 nucleotides, less than4 nucleotides, less than 3 nucleotides, less than 2 nucleotides or lessthan 1 nucleotide. It is preferred that they do not overlap at all. Stemprimers may further still be defined as primers that bind the region 3′of the forward primer binding region on the forward strand and 3′ of thereverse primer binding site on the reverse strand but where the primerbinding regions do not substantially overlap with any intra-molecularsecondary structure generated as a direct consequence of the primersemployed by a particular NAAT, especially a LFP.

The use of Stem primers significantly increases the rate ofamplification. This has the distinct advantage that diagnostic tests,for example, can deliver test results in a shorter period of time,something of common value amongst users of diagnostic tests. Anadditional benefit of faster amplification is that it can decrease thepossibility of false positive results and hence increase the specificityof a test. It has been observed that NAATs employing strand displacingpolymerases become increasingly prone to non-specific amplification asthe length of time required for amplification increases. As such, fasteramplification can also lead to more accurate results. Therefore, the useof stem primers increases the rate of amplification of NAATs such asLoop-mediated Isothermal Amplification (LAMP), and provides greaterflexibility in primer selection for a given target template.

Thus, in another embodiment, the described methods include theperformance of an amplification technique based on the isothermalapproach used in LAMP and LAMP-STEM. The general approach forloop-mediated isothermal amplification (LAMP) is illustrated in FIG. 1.Briefly, forward and reverse loop-forming primers (known as FIB and BIPprimers) comprise, e.g., F1c and F2, B1c and B2, priming sites,respectively, on a target (single-stranded) nucleic acid template (FIG.1a-b ). The target nucleic acid can be from genomic DNA, cDNA, RNA. Itis sometimes necessary to denature (via heat, ionic strength, or solventconditions) double-stranded nucleic acid template to allow the primersto bind to the template.

In certain methods as described herein, a first-stage amplification(i.e., “pre-amp”) reaction, e.g., LAMP reaction, is performed. The LAMPreaction, which includes, e.g., the loop-forming primers and one or moredisplacement primers proceeds at a relatively slow rate. The LAMPreaction results in the formation of a single stranded amplicon referredto as a “dumbbell” comprising a stem or middle region flanked by loopson the 3′ end and 5′ end created by the loop-forming primers (Figurelc). Further amplification cycles result in the production ofconcatamers of the “dumbbell” amplicons having alternatingcomplementarity such that the loop-forming primers as well as thedumbbell structure drives additional rounds of extension.

In an exemplary amplification method as described herein, a first stageisothermal amplification reaction (i.e., “pre-amp step”) using a NAAT,for example, a LAMP method is performed (i.e., FIG. 1a-c ), whichincludes forward and reverse primers, FIP and BIP primers (comprisingF1c and F2, B1c and B2, priming sites, respectively), and displacementprimers (F3 and B3) to amplify a region on the target nucleic acidcomprising one or more SNP sequences. The reaction mixture, whichincludes, e.g., the LAMP—generated amplicon, buffer and amplificationreagents (e.g., dNTPs), and loop-forming primers can be utilizeddirectly in the second stage of an exemplary amplification method asdescribed herein.

In any of the embodiments described herein, the method further comprisesthe addition of at least one displacement or bumper primer in thefirst-stage and/or second-stage amplification reaction.

The method of synthesizing or amplifying nucleic acid according to thepresent invention is supported by the DNA polymerase catalyzing thestrand displacement-type reaction for synthesis of complementary chain.During the reaction, a reaction step not necessarily requiring thestrand displacement-type polymerase is also contained. However, forsimplification of a constitutional reagent and in an economicalviewpoint, it is advantageous to use one kind of DNA polymerase. As thiskind of DNA polymerase, the following enzymes are known. Further,various mutants of these enzymes can be utilized in the presentinvention insofar as they have both the sequence-dependent activity forsynthesis of complementary chain and the strand displacement activity.The mutants referred to herein include those having only a structurebringing about the catalytic activity required of the enzyme or thosewith modifications to catalytic activity, stability or thermostabilityby e.g. mutations in amino acids. Exemplary polymerases for use in thedescribed methods include, Bst DNA polymerase, Bca (exo-)DNA polymerase,DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (exo-)DNApolymerase (Vent DNA polymerase deficient in exonuclease activity), DeepVent DNA polymerase, Deep Vent(exo-)DNA polymerase (Deep Vent DNApolymerase deficient in exonuclease activity), .PHI.29 phage DNApolymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase (TakaraShuzo Co., Ltd.), KOD DNA polymerase (Toyobo Co., Ltd.).

Among these enzymes, Bst DNA polymerase and Bca (exo-) DNA polymeraseare particularly desired enzymes because they have a certain degree ofthermostability and high catalytic activity. The reaction of thisinvention can be carried isothermally in a preferred embodiment, butbecause of the adjustment of melting temperature (Tm) etc., it is notalways possible to utilize temperature conditions desired for thestability of the enzyme. Accordingly, it is one of the desiredconditions that the enzyme is thermostable. Although the isothermalreaction is feasible, heat denaturation may be conducted to providenucleic acid as a first template, and in this respect too, utilizationof a thermostable enzyme broadens selection of assay protocol.

Vent (exo-) DNA polymerase is an enzyme having both strand displacementactivity and a high degree of thermostability. It is known that thecomplementary chain synthetic reaction involving strand displacement byDNA polymerase is promoted by adding a single strand binding protein(Paul M. Lizardi et al., Nature Genetics, 19, 225-232, July, 1998). Thisaction is applied to the present invention, and by adding the singlestrand binding protein, the effect of promoting the synthesis ofcomplementary chain can be expected. For example, T4 gene 32 iseffective as a single strand binding protein for Vent (exo-) DNApolymerase.

FIG. 2 illustrates an exemplary embodiment of the second-stageamplification reaction, a LAMP-STEM technique as described herein. Inthis example, the second-stage amplication reaction further comprises atleast one site-specific primer (i.e., a site-specific Stem primer),which anneals or is complementary to a site-specific Stem primer-bindingregion or sequence in the amplicon from stage-one. In a preferredembodiment, the site-specific Stem primer has at its 3′ end a nucleotidethat is complementary to a nucleotide of interest (e.g., a SNP) in theamplicon from stage-one.

In certain embodiments, the methods include monitoring and comparing therate of amplification in the second-stage reaction. If the site-specificStem primer binding region in the amplicon from stage-one contains anucleotide complementary to the 3′ end of the site-specific Stem primer,the template will be amplified at an enhanced rate relative to amismatch primer (i.e., a primer that binds to the same location as thesite-specific Stem primer but that has a nucleotide at its 3′ end thatis not complementary to the amplicon). Because of the differentialamplification rate between the SNP-Stem primer and a mismatch primer, itis possible to run both reactions in parallel and compare the rates. Themore rapid appearance of amplification products in the site-specificStem primer relative to the mismatch primer is indicative of thepresence of the allele of interest.

The ability of loop-forming primers to generate stable, single strandedregions of amplicon is important to rapidly propagating further ampliconand represents a key aspect of technologies employing these primers. Itmeans that concatameric amplicon can contain many new priming sites forthe primers employed by the NAAT in question. In, e.g., LAMP, theloop-forming primers, which generate inverted repeats in the amplicon,also provide for single stranded regions of amplicon which they canthemselves bind to and so initiate further re-copy of amplicon and hencefurther propagate amplification. In LAMP further additional primers maybe used in addition to LFPs, which also bind to these single-strandedregions of amplicon to help further propagate amplification (known asloop primers). A facet of the present invention is that the stem primersdo not bind to said stable single stranded loops generated byloop-forming primers and/or loop primers but accelerate amplificationvia a distinct mechanism.

The LAMP-STEM component of the technique as described herein can furtherinclude additional primers, for example, “Loop primers” (FIG. 3) thatanneal to regions within the loops generated by the Loop-formingprimers. Loop pimers can further enhance the rate of the LAMPamplification reaction. In any of the embodiments described herein, themethod further comprises the addition of at least one loop primer whichis complementary to a region in the first loop or the second loop of theamplicon.

In any of the embodiments described herein, first-stage amplificationreaction is preceeded by a heating step in which the target nucleic acidtemplate and primers are heated to approximately 95° C. for from about 1minute to about 30 mintues, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 ,14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 2,5 26, 27, 28, 29,30, including all times in between. In certain embodiments, the targetnucleic acid template and primers are heated to approximately 95° C. forfrom about 5 minutes to about 10 minutes.

In any of the embodiments described herein, the amplification reaction(i.e., first-stage and/or second stage) is performed at a temperature offrom about 50° C. to about 80° C. In certain embodiments, theamplification reaction is performed at a temperature of about 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80° C. In certain embodiments, the amplificationreaction is performed at a temperature of from about 55° to about 65° C.

In any of the embodiments described herein, the site-specific primer isin the forward direction. In certain embodiments, the site-specificprimer is in the reverse direction. In still additional embodiments,both forward and reverse site-specific primers are included in thereaction.

In any of the embodiments described herein, a site-specific nucleotideis comprised at the 3′ end of at least one of a loop-forming primer, adisplacement primer, a site-specific primer, a loop primer orcombination thereof.

In any of the embodiments described herein, the primers or nucleotidereagents comprise a chemical modification.

In accordance with the above, an exemplary two-stage isothermal nucleicacid amplification method for the detection of a site of interestcomprises the steps of:

-   -   A) performing a first-stage nucleic acid amplification reaction        comprising:        -   1) providing a target nucleic acid template which comprises            at least a first primer binding region, and a first and            second loop-forming primer, wherein the target nucleic acid            template defines a region of interest and comprises a site            of interest;        -   2) annealing a first loop-forming primer comprising a first            and a second segment to the target template, wherein the            first segment is substantially complementary to the first            primer binding region on the template and the second segment            comprises a sequence that is substantially complementary to            another region in the first primer or a region in the            amplicon generated from the first segment of the first            loop-forming primer such that the second segment is able,            under suitable conditions, to form a first single-stranded            loop in the amplicon;        -   3) extending the first loop-forming primer from its 3′ end            under suitable conditions with a polymerase having strand            displacement activity, to form a single-stranded nucleic            acid molecule, wherein the second segment of the first            loop-forming primer hybridizes to a region in the amplicon            generated from the first segment of the first loop-forming            primer to form a first single-stranded loop having the 5′            end of the first loop-forming primer;        -   4) annealing to the amplicon from (3) a second loop-forming            primer comprising a first and a second segment, wherein the            first segment is substantially complementary to a            second-loop forming primer binding region on the amplicon            from (3), and the second segment comprises a sequence that            is substantially complementary to another region in the            second loop-forming primer or a region in the amplicon            generated from the first segment of the second loop-forming            primer such that the second region is able to form a second            single-stranded loop in the amplicon;        -   5) extending the second loop-forming primer from its 3′ end            under suitable conditions with a polymerase having strand            displacement activity, to form a single-stranded nucleic            acid molecule, wherein the second segment of the second            loop-forming primer hybridizes to a region in the amplicon            to form a second single-stranded loop having the 5′ end of            the second loop-forming primer;        -   6) repeating steps A2-5, and thereby amplifying the nucleic            acid template region between the two loop-forming primers            comprising the site of interst, wherein a mixure is formed            comprising the amplified nucleic acid (primary amplicon),            and first and second loop-forming primers; and    -   B) performing a second-stage amplification reaction comprising:        -   1) providing the mixture from step A6, and separating into a            plurality ofreactions, wherein, to at least one of            thereactions, at least one site-specific Stem primer is            included, which is substantially complementary to a            site-specific primer binding region (site-specific region of            interest) in the primary amplicon, and wherein at least one            other reactionincludes no secondary primer;        -   2) annealing the Stem primer, and the first and second            loop-forming primers to theprimary amplicon, and extending            them from their 3′ ends under suitable conditions with a            polymerase having strand displacement activity;        -   3) repeating step B2, and thereby further amplifying the            primary amplicon; and        -   4) detecting and comparing in real-time the rate of            amplification by the site-specific Stem primer and rate of            amplification in the reaction with no secondary primer,            wherein an increased rate of amplification by the            site-specific Stem primer relative to other reaction is            indicative of the presence of the site of interest in the            amplified nucleic acid, and wherein a slower or similar rate            is indicative of the absence of the site of interest in the            amplified nucleic acid.

In certain embodiments, the second-stage amplification reaction proceedsat an increased rate relative to the first.

In certain embodiments, the plurality of reactions include approximatelyequal volumes or amounts of the primary amplicon from the first-stageamplification reaction.

In certain embodiments, step (B)(1) further comprises including to atleast one additional reaction a mismatch Stem primer. In additionalembodimetns, step (B)(4) comprises detecting and comparing in real-timethe amplification rate of the site-specific primer reaction, themismatch primer reaction, and the reaction with no secondary primer,wherein an increase in the amplification rate in the site-specificprimer reaction relative to the others (as well as the first-stagereaction) is indicative of the presence of the site of interest.

As would be appreciated by those of skill in the art, in any of theaspects or embodimens as described herein, the methods can be modifiedso as to include any desired number of individual or discretesecond-stage reactions, any number of which can include, respectively,no secondary primer, a site-specific primer or a mismatch primer.Indeed, the methods include configurations where the same or differentsite-specific primers and/or the same or different mismatch primers arerun in parallel in separate reactions, and all are detected and comparedsimultaneously and in real-time.

Where the primers further contain a second segment, the second segmentcomprises a sequence that is substantially complementary to anothersegment in the first primer or a region in the amplicon generated fromthe first segment of the first primer such that the second region isable to form a loop. “An amplicon generated from the first segment ofthe first primer (or second primer)” refers to the first copy of thetemplate which is generated when the first primer is extended by apolymerase. Said amplicon includes the sequence of the first primer atits 5′ end.

In certain embodiments, the second segment is substantially identical toa region on the target template and/or the amplicon to which the primerbinds. Such primers are referred to herein as loop-forming primers.“Substantially identical” means that the second segment has at least70%, 80%, 90%, 95%, 99% or 100% identity to the region on the targettemplate and/or the amplicon. It is also envisioned that only part ofthe second region shows substantial identity with a region on the targettemplate. Regardless of whether the whole or only part of the secondsegment of the primer shows substantial identity with a region on thetarget template, the region of the second segment which is substantiallyidentical to a region on the target template and/or amplicon is at least5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, atleast 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides,at least 60 nucleotides, or even at least 70 nucleotides in length. Inthis aspect of the invention, once the first segment of the primer hasbeen extended to form a first amplicon, the second segment is able tobind to a complementary region within the same strand and thereby form aloop.

The methods of the invention may be practised using forward and reverseprimers of the same kind, e.g. loop-forming primers (LFP), hairpinprimers, etc. When referring to “the same kind of primers”, it is meantthat the primers are all simple primers, LFPs, LPPs or hairpin primers.The term “different kind of primers” accordingly relates to acombination of two or more primers wherein at least one of the primersis not of the same kind as the other primer(s). For example, where amethod uses four primers of which three are LFPs and one is a LPP, theprimers would be considered to be of a different kind. Thus, it is alsoenvisioned to use forward and reverse primers which are not of the samekind. For example, a forward primer may be used that is a LFP incombination with a reverse primer that is a LPP or a hairpin primer. Itis also possible to combine LFPs or hairpin primers with simple primersprovided that the combination of primers results in the formation of aconcatamer. Where the NAAT used for amplification employs more than one(i.e. two or more) forward and/or reverse primer, it is also possible tocombine the same or different kinds of primers on the same primerbinding site. In one aspect of the present invention, the two or moreforward and/or reverse primers are all LFPs. Suitable combinations ofprimers will be evident to those of skill in the art. For example, itwill be evident to the skilled person that the combination of forwardand reverse primers that are all simple primers may not provide amechanism to provide for the formation of a concatamer and thereforesuch a combination is not suitable for use in the present invention.

It is to be understood that, in general, the forward and reverseprimers, or sets of primers, act on different strands of the targettemplate. Furthermore, the primers (or one of each set of primers) willact to delimit the region of the original polynucleotide copied andrecopied. Thus exponential amplification requires the coupling ofactivities between at least two primer binding regions, a forward primerbinding region and a reverse primer binding region (FIG. 1). The forwardand reverse primer binding regions may each comprise a single bindingsite for a primer whereby the sites are on opposite sense strands i.e.one primer binding site is on the “forward strand”, one on the “reversestrand” (as shown in FIG. 1). The forward and reverse primer regions mayalso comprise binding sites for two or more primers each, where morethan two primers are employed by a particular NAAT. In this case, it ispossible that the two or more primer binding sites in the forward and/orreverse primer binding regions are all situated on the same strand ofthe target template and/or amplicon or on different strands of thetarget template and/or amplicon.

Stem primers as used in certain methods described herein may bepositioned anywhere between the forward and reverse primer bindingregions provided that the binding site(s) of the stem primer(s) do(es)not significantly overlap with the forward or reverse binding site. Itis to be understood that in the case where a LFP is employed, where theLFP is a forward primer, the forward primer binding region encompassesnot only the F2 site (i.e. the forward primer binding region) but alsothe F1 site (i.e. the region on the forward strand which issubstantially identical to the second segment of the LFP), and where theLFP is a reverse primer, the reverse primer binding region encompassesnot only the R2c site (i.e. the reverse primer binding region but alsothe Rlc site (i.e. the region on the reverse strand which issubstantially identical to the second segment of the LFP; FIG. 1). Inthis way the stem primers may be positioned anywhere between the R1(c)and F1(c) sites where two LFPs are employed (as in LAMP and TRA); wherea single LFP is employed in a particular NAAT, the stem primers may bindbetween either a R1(c) or F1(c) site and another primer binding regionoccupied by a non-LFP.

It is possible to employ only one stem primer which binds either theforward or reverse polynucleotide strand (as shown in FIG. 2).Alternatively, two or more stem primers may be used which can bindeither to strand of the amplicon or to the same strand. The methods ofthe present invention may be practised with one, two, three, four ormore stem primers which can be used in any spatial combination and whichmay bind either the reverse or forward strand provided that the bindingsites for the stem primers do not significantly overlap with the forwardor reverse primer binding regions or do not overlap at all. The stemprimers may further bind to any part within the stem region. Thus, thestem primer(s) may have a binding site which is in close proximity tothe forward or reverse primer binding region. “Close proximity” meansthat the binding region of the stem primer and the forward/reverseprimer binding region are no more than 10 bp, 50 bp, 100 bp, 200 bp, 300bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp or 1000 bp apart.

In certain embodiments, the stem primers may be at least 5 nucleotides,at least 10 nucleotides, at least 20 nucleotides, at least 30nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least60 nucleotides, at least 70 nucleotides, at least 80 nucleotides or atleast 90 nucleotides in length. The stem primers may be simple primers.However, it is also envisioned to use stem primers that are LFPs,hairpin primers, LPPs, chimeric primers, or other derivatives. Wheremore than one stem primer is used, the stem primers may be of the samekind or may be a combination of different kinds of primers.

There is a great variety of possible combinations of “simple primers”,LFPs, hairpin primers, RNA containing primers, nickase site containingprimers and other novel primers which could be used in novelcombinations to generate derivatives of the methods outlined inrespective NAAT methods. Where said combinations result in methods whichgenerate concatameric amplicon capable of self-copying to generatelonger concatamers, stem primers are anticipated to be applicable.

In certain embodiments, the site-specific Stem primer comprises at its3′ end a nucleotide that is complementary to a nucleotide of interest inthe amplified nucleic acid template or amplicon. In still additionalembodiments, the mismatch Stem primer comprises a 3′ end nucleotide thatis not complementary to a nucleotide of interest in the amplifiednucleic acid template or amplicon.

In certain additional embodiments, the complementary or site-specificStem primers may decrease the time required to detect a particular typeand amount of target template by at least 1 minute, at least 2 minutes,at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 20minutes, at least 30 minutes or at least 60 minutes compared to acontrol reaction to which no stem primer(s) or a mismatch stem primerhas/have been added.

Stem primers act to increase the rate of amplification of methodsemploying LFPs via the coupling of processes occurring at the forwardand reverse binding regions and since it has been taught in theliterature that the LAMP method has an upper limit to the number ofnucleotides separating the forward and reverse binding sites for theLFPs employed (Notomi et al. Loop-mediated isothermal amplification ofDNA, Nucleic Acids Research, (2000) Vol 28., No. 12, e63), the use ofstem primers can clearly allow the forward and reverse binding sites tobe located further apart in the sequence than previously practicable(especially if several stem primers are employed). This can have greatbenefit when it is desirable to demonstrate that two regions of sequenceoccur together on a polynucleotide but where the distance between thetwo regions is too far to allow each respective region to be effectivelyused as a forward and reverse binding region in the NAATs describedherein.

Since use of stem primers can allow for the forward and reverse bindingregions to be much further apart than in their absence and still allowfor effective amplification, the presence of two distinct sites on apolynucleotide can be established. Thus the invention provides a methodfor amplification of a polynucleic acid wherein the forward and reverseprimer binding regions are located at a distance such that synthesis ofa polynucleic acid can occur only in the presence of the stem primer(s).This distance can be defined experimentally by performing two separateNAATs in parallel wherein the NAATs, the reagents and the amplificationconditions used are identical except that stem primer(s) are added toone reaction but not the other. Where synthesis of the polynucleic acidoccurs only in the presence of the stem primer(s), the primer bindingsites are considered to be located at a distance such that synthesis ofa polynucleic acid can occur only in the presence of the stem primer(s).

The stem primers may contain exclusively naturally occurring nucleicacids. However, it is also envisioned to utilise primers that containmodified bases. Examples of such modified bases include, but are notlimited to, N4-methylcytosine, inosine, ribocleotides, fluorescentbases, photolysable bases or universal bases. It is also envisioned touse nucleic acids that have been labelled with a moiety that allows thestem primer and/or the amplicon to which the labelled stem primer bindsto be detected. For example, the nucleic acid may be fluorescentlylabelled. The stem primers may alternatively be labelled with capturemoieties (e.g. biotin).

Importantly, the stem primers are not directly responsible forexponential amplification of the amplicon, which is mediated by theprimers binding to the forward and reverse primer binding sites, butmerely increase the rate of amplification. This is because the stemprimers are considered to function on the amplification products of theother primers employed by a particular NAAT. Hence, stem primersfunction by increasing the amplification rate of the reaction mediatedby the forward and reverse primers. This is shown in FIG. 2, where itcan be seen that were the stem primer to prime and extend from thetarget template, the partial copy of the target template would containonly either the forward primer binding region or the reverse primerregion, but not both. Therefore, the principal amplicon generated from astem primer would not allow for reciprocal copying and hence would notcontribute to exponential amplification of the target template. The sameargument applies to stem primers copying a principal amplicon generatedby other primers employed by a particular NAAT and similarly for firstgeneration amplicons.

Also, stem primers are only anticipated to significantly increase therate of amplification of a target template if the next generationamplicon (i.e. further copies of the first generation amplicon (andcopies of these copies)) is concatameric in nature. The requirement forstem primers to work on concatamers follows from the requirement thatfor a particular polynucleic acid to contribute to exponentialamplification it must contain regions capable of acting as the forwardand reverse primer binding regions. Copying of a concatameric structurevia a stem primer, can produce a polynucleotide copy which has bothforward and reverse primer binding sites, whereas copying anon-concatameric structure does not. Thus, the use of stem primers willbe beneficial for amplification methods that result in the formation ofconcatamers.

The methods as described herein are well-suited for use with isothermalnucleic acid amplification methods making them relatively easy toperform, efficient, robust and reproducible. An advantage of thedescribed methods is that they allow for the rapid identification of adesired target nucleic acid (e.g., genetic mutation, SNP, pathogenicinfection, etc.) in a sample, including a complex mixture. The rapidamplification necessarily means that less time is required to identifythe presence or absence of a targeted genetic element, and therefore,the present methods provide a convenient means for point-of-care geneticdetection and diagnosis.

The term “substantially complementary” means that the first segment hassufficient complementarity to bind to the primer binding region on thetemplate and/or amplicon under conditions which are commonly used duringNAATs. This requires that the first segment of the primer has at least70%, 80%, 90%, 95%, 99% or 100% complementarity to the primer bindingregion on the template. The first segment of the primer may be at least5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, atleast 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides,at least 60 nucleotides, or even at least 70 nucleotides in length.

In any of the embodiments described herein, the first or second-stageprimers can comprise an endonuclease restriction site, or recognitionelement for a nicking enzyme.

In any of the embodiments described herein, the amplification reactioncan comprise a hairpin primer comprising a first and a second segment,wherein the first segment is substantially complementary to a primerbinding region on a template and the second segment comprises a sequencethat is substantially complementary to another region in the primer;

In any of the embodiments described herein, the amplification reactioncan comprise a loop-providing primer, comprising a hairpin primer inwhich the inverted repeats are separated by a linker region.

In any of the embodiments described herein, the amplification reactioncan comprise a chimeric primer.

In any of the aspects or embodiments described herein, the first-stageamplification reaction may be performed in a container or chambercomprising a channel that is in fluid communication with one or moreadditional containers or chambers comprising primers for performing thesecond-stage amplification reaction (i.e., site-specific primer,site-specific Stem primer; mismatch primer, mismatch Stem primer, etc).In still additional embodiments, the first-stage reaction chambercomprises a channel that is in one-way fluid communication with one ormore additional containers or chambers comprising primers for performingthe second-stage amplification reaction. For example, as illustrated inFIG. 4 the first-stage reaction is performed in a central chamber thatis in fluid communication with a plurality of additional chambers forperforming the second-stage amplification reaction. In a preferredembodiment, the reaction mixture from the first-stage amplificationreaction is automatically and simultaneously introduced into theadditional chambers, which comprise the primers for performing thesecond-stage reaction.

In a preferred embodiment, an approximately equal amount or volume ofthe first-stage reaction mixture is automatically introduced from acentral chamber to the axially arranged additional chambers via, e.g.,centrifugal force. In certain embodiments, the method comprisesperforming a plurality of separate or discrete second-stageamplification reactions approximately simultaneously. In certainadditional embodiments, the discrete reactions comprise the same ordifferent site-specific primers. In still additional embodiments, thediscrete reactions comprise a plurality or combination of site-specificprimers (i.e., “multiplexing reaction”).

In any of the aspects or embodiments described herein, each second-stagereaction volume can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 1000 or more. In apreferred embodiment, each respective second-stage reaction volume is 8μl.

In certain embodiments, the description provides a method for detectingand comparing nucleic acid amplification comprising providing afirst-stage reaction chamber; performing a first-stage pre-amplificationreaction as described herein in the first-stage reaction chamber;introducing an amount or volume of the first-stage pre-amp reactiondirectly into a plurality of second-stage reaction chambers, wherein atleast one second-stage reaction chamber comprises a site-specificprimer, and at least one other second-stage reaction chamber comprises amismatched primer; and performing in each second-stage reaction chambera second-stage amplification reaction as described herein; and detectingand comparing in real-time the rate of amplification of each reactionsimultaneously, wherein a faster rate of amplification in thesite-specific primer reaction versus the mismatch primer reaction isindicative of the presence of the site of interest.

In any of the aspects or embodiments described herein, the first-stageamplification reaction can comprise site-specific primers such that thefirst-stage pre-amplification reaction is selective for a particularsite of interest, and only if that site or region exists in the samplewill rapid amplification of the template proceed at the second or laterstage amplification reaction.

In any of the aspects or embodiments described herein, the second-stage(or later stage) amplification reaction can comprise multiplesite-specific primers (i.e., “multiplex reaction”). By usingdifferentially labeled site-specific oligonucleotides differentamplification products can be detected and compared (i.e., multiplexed)within the same reaction.

Although several of the aspects and embodiments refer to a two-stagedamplification scheme, the invention is not so limited. Indeed, themethods described herein are predicated upon the surprising discoverythat the presence of a specific site or sequence of interest in a targetnucleic acid region can be detected by the enhanced rate ofamplification with a site-specific or sequence-specific (i.e.,complementary) primer relative to the slower rate of an amplificationreaction with mismatch primer that, but-for the mismatched base, annealsto the same target region. As such, any number of additionalamplification steps can be performed, e.g., 3, 4, 5, 6, 7, 8, 9, etc.,and therefore, any number of sites, regions or sequences of interest canbe detected. Similarly, for each of the respective site-specific primerreactions to be performed, a parallel mismatch primer reaction can beperformed and the rate of amplification of the two reactions monitored.

The skilled person will be aware that, in addition to the primers neededfor amplification, the NAATs will require further reagents in order tosynthesize a polynucleic acid. The required reagents will be evident tothe person skilled in the art but will generally include a suitablebuffer, dNTPs, a polymerase, etc.

As the skilled person will appreciate, following addition of all thenecessary components for performing the NAAT in question, it isnecessary to provide suitable conditions for the synthesis of thepolynucleic acid. This can be achieved by providing a suitableincubation temperature, for example. It is preferred that amplificationoccurs under isothermal conditions. This means that during amplificationthe temperature is kept constant. “Constant” means that the temperaturevaries by no more than +/−10° C. However, methods that encompass asingle temperature change of greater than 10° C., two temperaturechanges of greater than 10° C., three temperature changes greater than10° C., four temperature changes greater than 10° C. or five temperaturechanges greater than 10° C. during the amplification process are alsowithin the scope of the present invention.

In another aspect, the description provides a two-stage isothermalnucleic acid amplification and detection method comprising the steps of:

-   -   a. providing a LAMP amplified nucleic acid template comprising        concatamerized amplicons, wherein the amplicons are formed        of (i) a 3′ end portion comprising a first region located 3′        terminal that, under suitable conditions, anneals to a first        complementary region to form a first loop; and (ii) a 5′ end        portion comprising a second region located 5′ terminal that,        under suitable conditions, anneals to a second complementary        region to form a second loop;    -   b. admixing a portion of the nucleic acid template from (a)        to: (i) a reaction mixture comprising at least one site-specific        Stem primer that is substantially complementary to a site or        region of interest between the first and second primer binding        regions, wherein the site-specific primer includes at its 3′ end        a nucleotide that is complementary to a nucleotide of interest        in the template, and (ii) a reaction mixture comprisingno        secondary primer;    -   c. annealing the primers from (b) to the nucleic acid template;    -   d. extending the primers from the 3′ end under suitable        isothermal conditions with a polymerase having strand        displacement activity; and    -   e. repeating steps c-d, and thereby rapidly amplifying the        nucleic template, and detecting and comparing in real-time the        amplification rate of reaction (b)(i) with reaction (b)(ii),        wherein an increase in the amplification rate in reaction (b)(i)        relative to reaction (b)(ii) is indicative of the presence of        the site of interest.

In certain embodiments, each reaction mixture includes approximately anequal volume or amount of the nucleic acid template.

In certain embodiments, step (b) further comprises admixing a portion ofthe nucleic acid template from (a) to (iii) a reaction mixturecomprising a mismatch Stem primer. In additional embodimetns, step (e)comprises detecting and comparing in real-time the amplification rate ofreactions (b)(i), (b)(ii), and (b)(iii), wherein an increase in theamplification rate in reaction (b)(i) relative to reaction (b)(ii) and(b)(iii) is indicative of the presence of the site of interest.

In any of the aspects or embodiments described herein, the methodfurther comprises a step of detecting the formation of an amplicon inreal-time. In certain embodiments, the amplification reaction mixturecomprises fluorescently labeled dNTPs, a fluorescent DNA intercalatingdye, chemiluminescent, electrochemical or other reporter system as ameans to follow the extent of amplification in real-time. Thus, incertain embodiments, the formation of an amplicon is detected inreal-time by measuring an increase in fluorescence. The amplification ofthe polynucleic acid according to the invention may be detected bymethods known to those of skill in the art. Suitable methods include butare not limited to the use of fluorescent intercalating dyes,fluorescent primers or probes, measuring turbidity, electrochemicalprobes, bioluminescent signals and chemiluminescent probes.

The amplification of the polynucleic acid may be detected usingreal-time methods, i.e. methods that can detect the polynucleic acid asit is amplified. Examples of such detection systems include, but are notlimited to, fluorescence (e.g. fluorescent probes that are added duringthe amplification), bioluminescent signals and electrochemical probes.In one aspect, the primers themselves are labelled with a detectablemoiety, e.g. a fluorescent label, a chemiluminescent label or anelectrochemical label, that allows detection of the amplicon to whichthe primer(s) bind(s). Thus, a further utility of stem primers inconcatamer forming NAATs could be as probe for use in a fluorescent,chemiluminescent or electrochemical reporter system as a means to followthe extent of amplification in real-time. Other suitable reportersystems will be evident to those of skill in the art. Stem primers couldhave benefit as probe containing primers over e.g. LFP or hairpinprimers since they are not required to generate inverted repeats inamplicon which could affect certain types of probes. Alternatively, theamplification product may be detected using end-point measurements, i.e.measurements which take place after the amplification of the polynucleicacid has been completed.

The amplification of the polynucleic acid can also be detected by otherdetection methods employed in NAAT detection. Suitable examples include,but are not limited to, gene arrays, lateral flow strips,electrophoresis, mass spectroscopy and acoustic detection.

In one embodiment the Bioluminescent Assay in Real-Time (BART) reportersystem is used to detect the synthesis of the polynucleic acid. Thissystem has been explained in detail in WO2004/062338 and WO2006/010948,which are hereby incorporated by reference. BART is an example of areporter system designed for isothermal NAATs which gives a single typeof signal from a sample: a bioluminescent signal. BART utilizes thefirefly luciferase-dependent detection of inorganic pyrophosphate: thisis produced in large quantities when ‘target’ sequences are amplifiedusing a NAAT. As such, molecular diagnostics can be achieved with BARTsimply by measuring the light emitted from closed tubes, in ahomogeneous phase assay. BART is proven with several different NAATs,operating between 50-63° C. The BART reporter is a particularlyeffective means to follow the rate of amplification of a NAAT since thelight output represents a measure of the instantaneous rate ofamplification (whereas, e.g. fluorescent outputs show the accumulationof a signal and hence the measurements have to be differentiated toobtain the amplification rates).

BART being used in conjunction with LAMP to detect a dilution series ofa particular target DNA molecule. Note that as the amount of target DNAin the sample decreases, the lag-phase to reach the time of maximallight increase (which is proportional to the lag-phase to reach maximalamplification) increases. Put differently, the time to reach thecharacteristic light peak associated with positive samples in BARTincreases in inverse proportion to the amount of target polynucleic acidin the sample. It is stressed that whilst the examples make use of theBART reporter system, the present invention is not limited to the use ofBART and is equally applicable to methods such as fluorescence,turbidity, other spectroscopic techniques or electrochemical measurementmethods irrespective of whether these are employed in real-timemeasurement of amplification or as end-point measurements.

Preferably, the method of the invention is performed in a sealed vessel.This is of great utility since it reduces or even prevents thepossibility of the sample becoming contaminated. Moreover, it reduces oreven prevents the possibility of the laboratory becoming contaminated.This is particularly important as if even one copy of the templatepolynucleic acid or amplicon were to escape into the laboratory, thiscould potentially contaminate other samples to be tested and givefalse-positive results. Thus, the ability to prevent contamination is ofparticular importance where a method of the invention is used in adiagnostic application.

In another aspect, the description provides methods for determiningwhether a particular polynucleic acid sequence is present in a nucleicacid sample or in organism's genetic code. For example, the methods canbe used for determining whether the nucleic acid to which the templatenucleic acid originates has been genetically modified, for detection ofDNA associated with a particular non-genetically modified breed of plantor a genetically modified plant, for detection of DNA associated withpedigree breeds of animal or for medical or veterinary diagnosticapplications such as genetic testing or forensic.

In any of the embodiments described herein, the target nucleic acidtemplate can comprise genomic DNA, cDNA or RNA or a segment therefrom,from a virus, plant, microbe, fungus, mycoplasma, single cellularorganism, or multicellular organism, e.g., a mammal, such as a human. Incertain embodiments, the genomic DNA is from a pathogenic virus ormicrobe, e.g., bacteria or mycoplasma.

Thus, in another aspect, the description provides methods for diagnosticapplications. In particular the methods allow identification andquantification of organisms in a patient and other samples. The methodsof the present invention are also suitable for the detection ofmutations, genetic diseases, or single-nucleotide polymorphisms (SNPs).The methods of the present invention are also suitable for the detectionof pathogenic and non-pathogenic micro-organisms or viruses.

The organism may be any micro-organisms, such as viruses, bacteria,mycoplasma and fungi. The micro-organism can be pathogenic but it mayalso be a non-pathogenic micro-organism. The microorganism may also be agenetically modified organism (GMO). Furthermore, the methods of thepresent invention can be used to identify genetically modified crops andanimals, for the detection of a disease state; for the prediction of anadverse reaction from a therapy and also for the prediction of a diseasestate susceptibility.

For example, in one embodiment, the description provides a two-stagemethod of detecting the presence of a nucleic acid of interest ordiagnosing a disease. In certain embodiments, the method as describedherein can be used for the real-time, rapid identification of amicro-organism, such as a virus, bacteria, mycoplasma, fungus, or agenetic disease, mutation, SNP, wherein said method comprises providinga nucleic acid sample from subject to be tested, e.g., a patient sample,performing the two-stage amplification reaction as described herein, anddetecting and comparing in real-time the amplification rate of asite-specific primer amplification rate with a mismatch primeramplification rate, wherein an increase in the site-specific primeramplification rate relative to the mismatch primer amplification rate isindicative of the presence of the specific site, region or nucleotide(s)of interest, e.g., a site, region, or nucleotide(s) associated with atleast one of a genetic disease, mutation, SNP, virus, or microbe, e.g.,bacteria.

In certain embodiments, the microbe is a bacterium. In certainembodiments, the bacteria is a member of a genus selected from the groupconsisting of Bacillus, Bartonella, Bordetella, Borrelia, Brucella,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Enterococcus, Escherichia, Francisella, Haemophilus, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Treponema, Ureaplasma, Vibrio, and Yershinia.

In certain embodiments, the bacteria is a member of the group consistingof Bacillus anthracis, Bacillus cereus, Bartonella henselae, BartonellaQuintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii,Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucellacanis, Brucella melitensis, Brucella suis, Campylobacter jejuni,Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Francisella tularensis,Haemophilus influenza, Helicobacter pylori, Legionella pneumophila,Leptospira inlerrogans, Leptospira santarosai, Leptospira weilii,Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonasaeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonellatyphimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum,Ureaplasma urealyticum, Vibrio cholera, Yersinia pestis, Yersiniaenterocolitica, and Yersinia pseudotuberculosis.

In certain embodiments, the target nucleic acid template is fromtubercle bacillus (MTB or TB). In certain additional embodimetns, thetarget nucleic acid template is from the rpoB gene from MTB. In stillfurther embodiments, the target nucleic acid template is rpoB13.5 F6.

In certain embodiments, the virus is a member of a family selected fromthe group consisting of Adenoviridae, Herpesviridae, Papillomaviridae,Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae,Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae,Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae,Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae.

In certain embodiments, the virus is a member selected from the groupconsisting of Adenovirus, Herpes simplex type 1, Herpes simplex type 2,Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Humanherpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox,Hepatitis B, Human bocavirus, Parvovirus B19, Human astrovirus, Norwalkvirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Severeacute respiratory syndrome virus, Hepatitis C virus, yellow fever virus,dengue virus, West Nile virus, Rubella virus, Hepatitis E virus, Humanimmunodeficiency virus (HIV), Influenza virus, Guanarito virus, Juninvirus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congohemorrhagic fever virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Humanmetapneumovirus, Hendra virus, Nipah virus, Rabies virus, Hepatitis D,Rotavirus, Orbivirus, Coltivirus, and Banna virus.

In a further aspect, there is provided a kit for use in a methodaccording to the invention. Preferably such a kit comprises all thecomponents necessary to practice a method as described herein, includingpaired site-specific and mismatch primers. In certain aspects the kitcomprises the target polynucleic acid which is to be tested, and/orreagents for preparing the biological sample for inclusion in themethods as described herein.

A kit for use in a method according to the invention preferablycomprises a polynucleic acid polymerase, the substrates for the nucleicacid polymerase and primers suitable for isothermal amplification of thetarget polynucleic acid, as described earlier. More preferably, the kitfurther comprises buffer reagents, such as a source of magnesium ions,or additives known in the art to improve the performance of a NAAT suchas Betaine or additives known to improve the shelf-life of kit reagentssuch as trehelose or additives known to help preserve reagents such assodium azide. Alternatively, a kit for use in a method according to theinvention may comprise only some of these components and/or additionalcomponents. The sample and any other components that have been omittedfrom the kit may then be added to the kit during use.

Preferably, at least one of the components of the kit is lyophilized oris in another form which is suitable for storage in the kit. Morepreferably, all of the components of the kit are lyophilized or in oneor more other forms suitable for storage. Such other forms includecomponents to which stabilizing factors have been added and/or arefrigerated or frozen master mix that contains the components of thekit.

In another aspect, the description provides a method of treating orpreventing a disease, comprising performing a two-stage nucleic acidamplification as described herein on a patient sample, and wherein ifthe patent tests positive for the presence of a site of interestassociated with a disease or infection, treating or administering to thepatient an effective amount of an appropriate therapeutic or bioactiveagent, e.g., antibiotic, anti-cancer agent, anti-inflammatory,antimicrobial, antiviral, antifungal, antipsychotic, etc. The term“bioactive agent” is used to describe an agent with biological activityto assist in effecting an intended therapy, inhibition and/orprevention/prophylaxis. The terms “treat”, “treating”, and “treatment”,etc., as used herein, refer to any action providing a benefit to apatient including the treatment of any disease state or condition.

Disease states of conditions which may be treated using compoundsaccording to the present invention include, for example, asthma,autoimmune diseases such as multiple sclerosis, various cancers,ciliopathies, cleft palate, diabetes, heart disease, hypertension,inflammatory bowel disease, mental retardation, mood disorder, obesity,refractive error, infertility, Angelman syndrome, Canavan disease,Coeliac disease, Charcot-Marie-Tooth disease, Cystic fibrosis, Duchennemuscular dystrophy, Haemochromatosis, Hemophilia, Klinefelter'ssyndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney disease,(PKD1) or 4 (PKD2) Prader-Willi syndrome, Sickle-cell disease, Tay-Sachsdisease, Turner syndrome.

Further disease states or conditions which may be treated by compoundsaccording to the present invention include Alzheimer's disease,Amyotrophic lateral sclerosis (Lou Gehrig's disease), Anorexia nervosa,Anxiety disorder, Atherosclerosis, Attention deficit hyperactivitydisorder, Autism, Bipolar disorder, Chronic fatigue syndrome, Chronicobstructive pulmonary disease, Crohn's disease, Coronary heart disease,Dementia, Depression, Diabetes mellitus type 1, Diabetes mellitus type2, Epilepsy, Guillain-Barré syndrome, Irritable bowel syndrome, Lupus,Metabolic syndrome, Multiple sclerosis, Myocardial infarction, Obesity,Obsessive-compulsive disorder, Panic disorder, Parkinson's disease,Psoriasis, Rheumatoid arthritis, Sarcoidosis, Schizophrenia, Stroke,Thromboangiitis obliterans, Tourette syndrome, Vasculitis.

Still additional disease states or conditions which can be treated bycompounds according to the present invention include aceruloplasminemia,Achondrogenesis type II, achondroplasia, Acrocephaly, Gaucher diseasetype 2, acute intermittent porphyria, Canavan disease, AdenomatousPolyposis Coli, ALA dehydratase deficiency, adenylosuccinate lyasedeficiency, Adrenogenital syndrome, Adrenoleukodystrophy, ALA-Dporphyria, ALA dehydratase deficiency, Alkaptonuria, Alexander disease,Alkaptonuric ochronosis, alpha 1-antitrypsin deficiency, alpha-1proteinase inhibitor, emphysema, amyotrophic lateral sclerosis Alströmsyndrome, Alexander disease, Amelogenesis imperfecta, ALA dehydratasedeficiency, Anderson-Fabry disease, androgen insensitivity syndrome,Anemia Angiokeratoma Corporis Diffusum, Angiomatosis retinae (vonHippel-Lindau disease) Apert syndrome, Arachnodactyly (Marfan syndrome),Stickler syndrome, Arthrochalasis multiplex congenital (Ehlers-Danlossyndrome#arthrochalasia type) ataxia telangiectasia, Rett syndrome,primary pulmonary hypertension, Sandhoff disease, neurofibromatosis typeII, Beare-Stevenson cutis gyrata syndrome, Mediterranean fever,familial, Benjamin syndrome, beta-thal assemi a, Bilateral AcousticNeurofibromatosis (neurofibromatosis type II), factor V Leidenthrombophilia, Bloch-Sulzberger syndrome (incontinentia pigmenti), Bloomsyndrome, X-linked sideroblastic anemia, Bonnevie-Ullrich syndrome(Turner syndrome), Bourneville disease (tuberous sclerosis), priondisease, Birt-Hogg-Dubé syndrome, Brittle bone disease (osteogenesisimperfecta), Broad Thumb-Hallux syndrome (Rubinstein-Taybi syndrome),Bronze Diabetes/Bronzed Cirrhosis (hemochromatosis), Bulbospinalmuscular atrophy (Kennedy's disease), Burger-Grutz syndrome (lipoproteinlipase deficiency), CGD Chronic granulomatous disorder, Campomelicdysplasia, biotinidase deficiency, Cardiomyopathy (Noonan syndrome), Cridu chat, CAVD (congenital absence of the vas deferens), Caylorcardiofacial syndrome (CBAVD), CEP (congenital erythropoieticporphyria), cystic fibrosis, congenital hypothyroidism, Chondrodystrophysyndrome (achondroplasia), otospondylomegaepiphyseal dysplasia,Lesch-Nyhan syndrome, galactosemia, Ehlers-Danlos syndrome,Thanatophoric dysplasia, Coffin-Lowry syndrome, Cockayne syndrome,(familial adenomatous polyposis), Congenital erythropoietic porphyria,Congenital heart disease, Methemoglobinemia/Congenitalmethaemoglobinaemia, achondroplasia, X-linked sideroblastic anemia,Connective tissue disease, Conotruncal anomaly face syndrome, Cooley'sAnemia (beta-thalassemia), Copper storage disease (Wilson's disease),Copper transport disease (Menkes disease), hereditary coproporphyria,Cowden syndrome, Craniofacial dysarthrosis (Crouzon syndrome),Creutzfeldt-Jakob disease (prion disease), Cockayne syndrome, Cowdensyndrome, Curschmann-Batten-Steinert syndrome (myotonic dystrophy),Beare-Stevenson cutis gyrata syndrome, primary hyperoxaluria,spondyloepimetaphyseal dysplasia (Strudwick type), muscular dystrophy,Duchenne and Becker types (DBMD), Usher syndrome, Degenerative nervediseases including de Grouchy syndrome and Dejerine-Sottas syndrome,developmental disabilities, distal spinal muscular atrophy, type V,androgen insensitivity syndrome, Diffuse Globoid Body Sclerosis (Krabbedisease), Di George's syndrome, Dihydrotestosterone receptor deficiency,androgen insensitivity syndrome, Down syndrome, Dwarfism, erythropoieticprotoporphyria Erythroid 5-aminolevulinate synthetase deficiency,Erythropoietic porphyria, erythropoietic protoporphyria, erythropoieticuroporphyria, Friedreich's ataxia., familial paroxysmal polyserositis,porphyria cutanea tarda, familial pressure sensitive neuropathy, primarypulmonary hypertension (PPH), Fibrocystic disease of the pancreas,fragile X syndrome, galactosemia, genetic brain disorders, Giant cellhepatitis (Neonatal hemochromatosis), Gronbl ad-Strandberg syndrome(pseudoxanthoma elasticum), Gunther disease (congenital erythropoieticporphyria), haemochromatosis, Hallgren syndrome, sickle cell anemia,hemophilia, hepatoerythropoietic porphyria (HEP), Hippel-Lindau disease(von Hippel-Lindau disease), Huntington's disease, Hutchinson-Gilfordprogeria syndrome (progeria), Hyperandrogenism, Hypochondroplasia,Hypochromic anemia, Immune system disorders, including X-linked severecombined immunodeficiency, Insley-Astley syndrome, Jackson-Weisssyndrome, Joubert syndrome, Lesch-Nyhan syndrome, Jackson-Weisssyndrome, Kidney diseases, including hyperoxaluria, Klinefelter'ssyndrome, Kniest dysplasia, Lacunar dementia,Langer-Saldinoachondrogenesis, ataxia telangiectasia, Lynch syndrome,Lysyl-hydroxylase deficiency, Machado-Joseph disease, Metabolicdisorders, including Kniest dysplasia, Marfan syndrome, Movementdisorders, Mowat-Wilson syndrome, cystic fibrosis, Muenke syndrome,Multiple neurofibromatosis, Nance-Insley syndrome, Nance-Sweeneychondrodysplasia, Niemann-Pick disease, Noack syndrome (Pfeiffersyndrome), Osler-Weber-Rendu disease, Peutz-Jeghers syndrome, Polycystickidney disease, polyostotic fibrous dysplasia (McCune-Albrightsyndrome), Peutz-Jeghers syndrome, Prader-Labhart-Willi syndrome,hemochromatosis, primary hyperuricemia syndrome (Lesch-Nyhan syndrome),primary pulmonary hypertension, primary senile degenerative dementia,prion disease, progeria (Hutchinson Gilford Progeria Syndrome),progressive chorea, chronic hereditary (Huntington) (Huntington'sdisease), progressive muscular atrophy, spinal muscular atrophy,propionic acidemia, protoporphyria, proximal myotonic dystrophy,pulmonary arterial hypertension, PXE (pseudoxanthoma elasticum), Rb(retinoblastoma), Recklinghausen disease (neurofibromatosis type I),Recurrent polyserositis, Retinal disorders, Retinoblastoma, Rettsyndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome, Roussy-Levysyndrome, severe achondroplasia with developmental delay and acanthosisnigricans (SADDAN), Li-Fraumeni syndrome, sarcoma, breast, leukemia, andadrenal gland (SBLA) syndrome, sclerosis tuberose (tuberous sclerosis),SDAT, SED congenital (spondyloepiphyseal dysplasia congenita), SEDStrudwick (spondyloepimetaphyseal dysplasia, Strudwick type), SEDc(spondyloepiphyseal dysplasia congenita) SEMD, Strudwick type(spondyloepimetaphyseal dysplasia, Strudwick type), Shprintzen syndrome,Skin pigmentation disorders, Smith-Lemli-Opitz syndrome, South-Africangenetic porphyria (variegate porphyria), infantile-onset ascendinghereditary spastic paralysis, Speech and communication disorders,sphingolipidosis, Tay-Sachs disease, spinocerebellar ataxi a, Sticklersyndrome, stroke, androgen insensitivity syndrome, tetrahydrobi opterindeficiency, beta-thalassemia, Thyroid disease, Tomaculous neuropathy(hereditary neuropathy with liability to pressure palsies), TreacherCollins syndrome, Triplo X syndrome (triple X syndrome), Trisomy 21(Down syndrome), Trisomy X, VHL syndrome (von Hippel-Lindau disease),Vision impairment and blindness (Alström syndrome), Vrolik disease,Waardenburg syndrome, Warburg Sjo Fledelius Syndrome,Weissenbacher-Zweymüller syndrome, Wolf-Hirschhorn syndrome, WolffPeriodic disease, Weissenbacher-Zweymüller syndrome and Xerodermapigmentosum, among others.

The term “cancer” refers to the pathological process that results in theformation and growth of a cancerous or malignant neoplasm, i.e.,abnormal tissue that grows by cellular proliferation, often more rapidlythan normal and continues to grow after the stimuli that initiated thenew growth cease. Malignant neoplasms show partial or complete lack ofstructural organization and functional coordination with the normaltissue and most invade surrounding tissues, metastasize to severalsites, and are likely to recur after attempted removal and to cause thedeath of the patient unless adequately treated. As used herein, the termneoplasia is used to describe all cancerous disease states and embracesor encompasses the pathological process associated with malignanthematogenous, ascitic and solid tumors. Exemplary cancers which may betreated by the present compounds either alone or in combination with atleast one additional anti-cancer agent include squamous-cell carcinoma,basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, andrenal cell carcinomas, cancer of the bladder, bowel, breast, cervix,colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas,prostate, and stomach; leukemias; benign and malignant lymphomas,particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign andmalignant melanomas; myeloproliferative diseases; sarcomas, includingEwing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas,astrocytomas, oligodendrogliomas, ependymomas, gliobastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas; bowel cancer, breast cancer, prostate cancer, cervicalcancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer,thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer,stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma,Hodgkin's disease, Wilms' tumor and teratocarcinomas. Additional cancerswhich may be treated using compounds according to the present inventioninclude, for example, T-lineage Acute lymphoblastic Leukemia (T-ALL),T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma,Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cellLymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia chromosomepositive ALL and Philadelphia chromosome positive CML.

The term “anti-cancer agent” is used to describe an anti-cancer agent.These agents include, for example, everolimus, trabectedin, abraxane,TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitor, an AKT inhibitor, an mTORC1/2 inhibitor, a JAK/STATinhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinaseinhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody,pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab,amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin,ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402, lucanthone, LY317615, neuradiab,vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin,ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide,gemcitabine, doxorubicin, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolyl-quinolone, vatalanib,AG-013736, AVE-0005, goserelin acetate, leuprolide acetate, triptorelinpamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate,megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide,megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib,canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016,Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanalide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248,sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide,L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, adriamycin,bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine,dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gleevec, gemcitabine,hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole,lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, teniposide,testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, vairubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox,gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS- 247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-0-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonist,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa, darbepoetin alfa and mixtures thereof.

The term “antivirals” include, for example, nucleoside reversetranscriptase inhibitors (NRTI), other non-nucleoside reversetranscriptase inhibitors (i.e., those which are not representative ofthe present invention), protease inhibitors, fusion inhibitors, amongothers, exemplary compounds of which may include, for example, 3TC(Lamivudine), AZT (Zidovudine), (−)-FTC, ddl (Didanosine), ddC(zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset), D4T(Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV(Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV(Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV (Nelfinavir), APV(Amprenavir), LPV (Lopinavir), fusion inhibitors such as T20, amongothers, fuseon and mixtures thereof, including anti-HIV compoundspresently in clinical trials or in development.

Other anti-HIV agents which may be used include, for example, otherNNRTI's (i.e., other than the NNRTI's according to the presentinvention) may be selected from the group consisting of nevirapine(BI-R6-587), delavirdine (U-90152S/T), efavirenz (DMP-266), UC-781(N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide),etravirine (TMC125), Trovirdine (Ly300046.HCl), MKC-442 (emivirine,coactinon), HI-236, HI-240, HI-280, HI-281, rilpivirine (TMC-278),MSC-127, HBY 097, DMP266, Baicalin (TJN-151) ADAM-II (Methyl3′,3′-dichloro-4′,4″-dimethoxy-5′,5″-bis(methoxycarbonyl)-6,6-diphenylhexenoate),Methyl 3-Bromo -5-(1-5-bromo-4-methoxy-3-(methoxycarbonyl)phenyl)hept-1-enyl)-2-methoxybenzoate(Alkenyldiarylmethane analog, Adam analog),(5-chloro-3-(phenylsulfinyl)-2′-indolecarboxamide), AAP-BHAP (U-104489or PNU-104489), Capravirine (AG-1549, S-1153), atevirdine (U-87201E),aurin tricarboxylic acid (SD-095345),1-[(6-cyano-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine,1-[5-[[N-(methyl)methylsulfonylamino]-2-indolylcarbonyl-4-[3-(isopropylamino)-2-pyridinyl]piperazine,1-[3-(Ethylamino)-2-[pyridinyl]-4-[(5-hydroxy-2-indolyl)carbonyl]piperazine,1-[(6-Formyl-2-indolyl)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine,1-[[5-(Methylsulfonyloxy)-2-indoyly)carbonyl]-4-[3-(isopropylamino)-2-pyridinyl]piperazine,U88204E, Bis(2-nitrophenyl)sulfone (NSC 633001), Calanolide A(NSC675451), Calanolide B,6-Benzyl-5-methyl-2-(cyclohexyloxy)pyrimidin-4-one (DABO-546), DPC 961,E-EBU, E-EBU-dm, E-EPSeU, E-EPU, Foscarnet (Foscavir), HEPT(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine), HEPT-M(1-[(2-Hydroxyethoxy)methyl]-6-(3-methylphenyl)thio)thymine), HEPT-S(1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine), InophyllumP, L-737,126, Michellamine A (NSC650898), Michellamine B (NSC649324),Michellamine F,6-(3,5-Dimethylbenzyl)-1-[(2-hydroxyethoxy)methyl]-5-isopropyluracil,6-(3,5-Dimethylbenzyl)-1-(ethyoxymethyl)-5-isopropyluracil, NPPS, E-BPTU(NSC 648400), Oltipraz(4-Methyl-5-(pyrazinyl)-3H-1,2-dithiole-3-thione),N-{2-(2-Chloro-6-fluorophenethyl]-N′-(2-thiazolyl)thiourea (PETT Cl, Fderivative),N-{2-(2,6-Difluorophenethyl]-N′-[2-(5-bromopyridyl)]thiourea {PETTderivative),N-{2-(2,6-Difluorophenethyl]-N′-[2-(5-methylpyridyl)]thiourea {PETTPyridyl derivative),N-[2-(3-Fluorofuranyl)ethyl]-N′-[2-(5-chloropyridyl)]thiourea,N-[2-(2-Fluoro-6-ethoxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea,N-(2-Phenethyl)-N′-(2-thiazolyethiourea (LY-73497), L-697,639,L-697,593, L-697,661, 3-[2-(4,7-Difluorobenzoxazol-2-yl)ethyl}-5-ethyl-6-methyl(pypridin-2(1H)-thione(2-Pyridinone Derivative),3-[[(2-Methoxy-5,6-dimethyl-3-pyridyl)methyl]amine]-5-ethyl-6-methyl(pypridin-2(1H)-thione,R82150, R82913, R87232, R88703, R89439 (Loviride), R90385, S-2720,Suramin Sodium, TBZ (Thiazolobenzimidazole, NSC 625487),Thiazoloisoindol-5-one, (+)(R)-9b-(3,5-Dimethylphenyl-2,3-dihydrothiazolo[2,3-a]isoindol-5(9bH)-one,Tivirapine (R86183), UC-38 and UC-84, among others.

Antimicrobial agents include, e.g., antibiotics. In certain embodiments,the anti-microbial is an anti-tuberculosis drug, e.g., pyrazinamide orbenzamide, pretomanid, and bedaquiline, among others.

EXAMPLES

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference. The above described compositionsand methods are further exemplified by reference to the Figures andaccompanying description below.

FIG. 6 illustrates the effect on rate of amplification due to mismatchedbases in Stem primers. (a) The left panel exemplifies a Stem primercomprising a 3′ end mismatch representing a primer that is not specificfor a SNP of interest. The 3′ mismatch induces a delay in theamplification reaction versus the matched or site-specific SNP Stemprimer (“SNP-Stem primer”). The increase in rate of amplification isexemplified in the right panel, wherein the dotted line represents theearliest time (t) to the point of maximum slope of fluorescence(t_(max)1), and ΔCt1 represents the lag in time to t_(max) between theSNP-Stem primer and the 3′ end mismatched Stem primer. (b) The rightpanel exemplifies another embodiment as described herein, wherein theStem primers (SNP-Stem primer and 3′ end mismatched primer) containanother base pair mismatch in their sequence. The second mismatch shiftsto the right (i.e., reduces the rate) of amplification (ΔCt2) and delaysthe time to t_(max)2.

FIG. 7 processed fluorescent traces showing matched Stem primer (i.e.,SNP-Stem primer) LAMP reactions (n=4), and mismatched Stem primer LAMPreactions (n=4) using the Bio-Rad CFX real-time PR instrument. Thereaction was held at 65° C. and each cycle consisted of a fluorescentmeasurement every 15 seconds. The reaction was assembled on ice in a 48well microplate. The solution consisted of about 200 copies of MTBgenomic DNA per reaction, 20 mM Tries pH 8.8, 10 mM ammonium sulfate, 50mM KCl, and 2.0 mM magnesium sulfate, 0.1% NP-40, 1× EVAgreen dye, BstDNA polymerase and 1.4 mM dNTPs.

FIG. 8 illustrates the enhanced rate of amplification observed accordingto an exemplary method as described herein. (a) Provides raw tracesshowing matched Stem primer reactions for site-specific Stem primerreactions (light traces) and mismatched Stem primer reactions (darktraces). (b) Quantification cycle (Cq; See Hellemans et al., 2007) ismeasured from the fitted raw traces as the point of maximum slope. TheCq for matched primers is plotted (light dots) along with mismatchedStem primers (dark dots) and no Stem primer LAMP (“dumbbell”) reactions(open circles). The cocktail for the first LAMP reaction (performed in afirst chamber) contained about 200 copies of MTB genomic DNA in asolution of AMP buffer (20 mM Tris pH 8.8, 10 mM ammonium sulfate, 50 mMKCl, and 3.4 mM magnesium sulfate, 0.1% NP-40, 1× EVAgreen dye, Bst DNApolymerase and 1.4 mM dNTPs). The solution was introduced into a firstchamber at 65° C., and incubated for 5 minutes before being distributeddirectly, automatically, and simultaneously into to a number of secondreaction chambers. LAMP primers to the rpoB gene were dried in the firstreaction chamber and supported LAMP amplification.

Accelerating Stem primers were dried in the second reaction chamber andwere either perfectly matched (representing a SNP-Stem primer) ormismatched at the 3′ end to the D435 drug resistance mutation of the MTBrpoB gene. As a negative control, several second reaction chamberscontained no Stem primers and demonstrated no accelerated amplification.Amplification in the second reaction chamber was detected by directlymeasuring the increase in fluorescence caused by intercalation ofEVAgreen dye into double-stranded amplified DNA. The fluorescencereaction was monitored continuously for 150 cycles of 15 secondsduration.

TABLE 1 rpoB STEM-LAMP Test. Matched Mismatched Dumbbell Δ(mismatched-Δ(dumbbell- Stem Cq Stem Cq Cq matched Stem) matched Stem) rpoB13.5 Unit2 38.5 ± 13.5% 66.8 ± 9.6% 72.3 ± 16.6% 14 (24% acc.) 4 (8% acc.) F6pre-amp (33~45) (59~72) (49~96) Unit 3 57.5 ± 9.1%  76.5 ± 1.3% 79.7 ±5.6%  13 (17% acc.) 6 (9% acc.) (50~62) (75~77) (68~86)

Exemplary Embodiments

In an embodiment, the description provides a two-stage nucleic acidamplification and real-time detection method comprising: a. providing acomposition comprising a target nucleic acid template and at least oneprimer that anneals to the target nucleic acid template near a region ofinterest to be amplified; b. performing a first nucleic acidamplification reaction to amplify the region of interest, therebyforming aprimary amplicon; c. dividing (b) into at least twosecondaryreactions, and including in at least one of the reactions asite-specific secondary primer that is complementary to a site-specificprimer binding site that may be present within the primary amplicon anddefines a site of interest within the region of interest; d. performinga second nucleic acid amplification reaction (second-stage reaction)thereby accelerating the amplification of the region of interest only ifthe site-specific primer binding site is complementary to thesite-specific primer; and e. detecting and comparing in real-time theamplification rates of the at least two secondary reactions, wherein anenhanced relative rate of amplification in the reaction with thesecondary primer indicates the presence of the sitee of interest that iscomplementary to the secondary primer.

In any of the embodiments described herein, step (a) includes a forwardand reverse loop-forming primer. In any of the embodiments describedherein, step (a) includes a forward and reverse displacement primer.

In any of the embodiments described herein, first nucleic acidamplification is an isothermal nucleic acid amplification reaction. Inany of the embodiments described herein, the second nucleic acidamplification is an isothermal nucleic acid amplification reaction.

In any of the embodiments described herein, the first and/or secondisothermal nucleic acid amplification reactions is LAMP or LAMP-STEM.

In any of the embodiments described herein, the primary amplicon is aconcatamer.

In any of the embodiments described herein, the site-specific primercomprises a 3′ end nucleotide complementary to a mutation, singlenucleotide polymorphism (SNP), allele or biomarker on the amplicon.

In any of the embodiments described herein, the method comprisesdividing (b) into at least an additional secondary reaction, wherein theadditional secondary reaction includes a mismatch secondary primer. Inany of the embodiments described herein, the mismatch primer comprises a3′ end nucleotide mismatch or 3′ end nucleotide complementary to anallele, mutation or polymorphism different from the site-specificprimer.

In any of the embodiments described herein, step (e) comprises detectingand comparing in real-time the amplification rates of all secondaryrections, wherein an enhanced relative rate of amplification as comparedto the other two reactions indicates the presence of the site ofinterest that is complementary to the secondary primer.

In any of the embodiments described herein, the mutation, SNP, orbiomarker is specific for a genetic disease, a microbe or a virus.

In any of the embodiments described herein, the step of performing thesecond nucleic acid amplification reaction includes amplification in thepresence of a fluorescent label or dye, thereby labeling theamplification products. In any of the embodiments described herein, thestep of detecting and comparing in real-time the amplification ratesincludes detecting and comparing a fluorescent signal from each of thereactions.

In any of the embodiments described herein, prior to the firstamplification reaction, the primer and template are heated to atemperature of approximately 95° C. for from about 1 minute to about 15minutes. In any of the embodiments described herein, the first andsecond amplification reactions are performed at a temperature of fromabout 55° C. to about 65° C.

In any of the methods described herein, the first amplification reactionis performed in a first reaction chamber including a channel that is inone-way fluid communication with a plurality of second reactionchambers, and wherein step (c) includes transporting approximately equalvolumes of (b) through the channels from the first reaction chamber tothe second reaction chambers.

In any of the embodiments described herein, the method of claim 1,wherein the relative reaction rates of the amplification reactionssatisfy the following condition: Second-stage amplificationreaction+site-specific primer>>first-stage amplificationreaction+mismatch primer >/≈first-stage amplification reaction.

In any of the embodiments described herein, the method of claim 1,wherein the Cq of the second-stage site-specific or complementary primeramplification reaction is at least 25% faster than the second-stagemismatch primer amplification reaction.

In another embodiment, the description also provides a two-stageisothermal nucleic acid amplification method for diagnosing or detectinga disease or infection method comprising: a. providing a compositioncomprising a nucleic acid sample from a patient, and at least one primerthat anneals to a target nucleic acid template near a region of interestto be amplified; b. performing a first isothermal nucleic acidamplification reaction to amplify the region of interest, therebyforming a primary amplicon; c. dividing (b) into at least tworeactions,and including to at least one of the reactions a site-specific secondaryprimer that is complementary to a site-specific primer binding regionthat may be present on the primary amplicon and defines a site ofinterest that is indicative of a disease or infection; d. performing asecond isothermal nucleic acid amplification reaction thereby amplifyingthe region of interest; e. detecting and comparing in real-time theamplification rate of thesecondary reactions, wherein an enhanced rateof amplification in the site-specific secondary primer reaction relativeto the other is indicative of the presence of the site of interest; andf. diagnosing the patient as having or not having a disease or infectioncorresponding to the site of interest.

In any of the embodiments described herein, the target nucleic acidtemplate is from a microbe or virus. In any of the embodiments describedherein, the target nucleic acid template is from tubercle bacillus (MTBor TB).

In any of the embodimetns described herein, the site of interest is inthe rpoB gene. In any of the embodiments described herein, the site ofinterest is a SNP in the rpoB gene.

In any of the embodiments described herein, the method comprises thestep of treating the patient testing positive for the site of interestby administering an effective amount of an appropriate therapeutic totreat the disease or infection. In any of the embodiments describedherein, the therapeutic is an anti-tuberculosis therapeutic.

In another embodiment, the description provides a two-stage nucleic acidamplification and real-time detection method comprising: a. providing acomposition comprising a target nucleic acid template and at least oneprimer that anneals to the target nucleic acid template near a region ofinterest to be amplified; b. performing a first nucleic acidamplification reaction to amplify the region of interest, therebyforming a primary amplicon; c. dividing (b) into at least threesecondary reactions, and including in at least one of the reactions asite-specific secondary primer that is complementary to a site-specificprimer binding site that may be present within the primary amplicon anddefines a site of interest within the region of interest, and includingin at least another of the reactions a mismatch secondary primer thatanneals at or near the site of interest but is not complementary to thesite of interest; d. performing a second nucleic acid amplificationreaction (second-stage reaction) thereby accelerating the amplificationof the region of interest only if the secondary primer binding site iscomplementary to the site-specific primer; and e. detecting andcomparing in real-time the amplification rates of the secondaryreactions, wherein an enhanced relative rate of amplification in thereaction with the site-specific secondary primer indicates the presenceof the site of interest that is complementary to the site-specificsecondary primer.

In any of the embodiments described herein, the method comprisesdividing (b) into at least an additional secondary reaction, wherein theadditional secondary reaction includes a mismatch secondary primer. Inany of the embodiments described herein, the mismatch primer comprises a3′ end nucleotide mismatch or 3′ end nucleotide complementary to anallele, mutation or polymorphism different from the site-specificprimer.

In any of the embodiments described herein, step (e) comprises detectingand comparing in real-time the amplification rates of all secondaryrections, wherein an enhanced relative rate of amplification as comparedto the other two reactions indicates the presence of the site ofinterest that is complementary to the secondary primer.

In any of the embodiments described herein, the method comprisesdividing (b) into at least one additional secondary reaction including asecond site-specific secondary primer complementary to a second site-ofinterest that may be present within the primary amplicon and defines asecond site of interest within the region of interest.Those skilled inthe art will recognize, or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to beencompassed by the following claims. It is understood that the detailedexamples and embodiments described herein are given by way of examplefor illustrative purposes only, and are in no way considered to belimiting to the invention. Various modifications or changes in lightthereof will be suggested to persons skilled in the art and are includedwithin the spirit and purview of this application and are consideredwithin the scope of the appended claims. For example, the relativequantities of the ingredients may be varied to optimize the desiredeffects, additional ingredients may be added, and/or similar ingredientsmay be substituted for one or more of the ingredients described.Additional advantageous features and functionalities associated with thesystems, methods, and processes of the present invention will beapparent from the appended claims. Moreover, those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1-34. (canceled)
 35. A method of detecting a pathogenic organism from abiological sample comprising a. providing a composition comprising atarget nucleic acid template from a pathogenic organism and acombination of site-specific primers that anneal to the target nucleicacid template near a region of interest to be amplified; b. performing afirst isothermal reaction to amplify the region of interest, therebyforming a primary amplicon; c. dividing (b) into at least two secondaryreactions, and including in at least one of the reactions one or moresite-specific secondary primer that is complementary to a site-specificprimer binding site that may be present within the primary amplicon anddefines a site of interest within the region of interest; d. performinga second isothermal reaction (second-stage reaction) therebyaccelerating the amplification of the region of interest only if thesite-specific primer binding site is complementary to the site-specificprimer; and e. detecting and comparing the amplification rates of the atleast two secondary reactions, wherein an enhanced relative rate ofamplification in the reaction with the secondary primer indicates thepresence of the site of interest in a pathogenic organism.
 36. Themethod of claim 35, wherein in step e), the detecting and comparing isin real time.
 37. The method of claim 35, wherein the pathogenicorganism detected is a virus, bacteria, archae, protozoa ormulticellular organism.
 38. The method of claim 35, wherein thepathogenic organism detected is a virus.
 39. The method of claim 35,wherein the pathogenic organism detected is a bacteria.
 40. The methodof claim 35, wherein the pathogenic organism detected is a coronavirus.41. The method of claim 36, wherein the amplification reaction mixturecomprises fluorescently labeled dNTPs, a fluorescent DNA intercalatingdye, bioluminescent, chemiluminescent, electrochemical or other reportersystem as a means to follow the extent of amplification in real-time.42. The method of claim 35, wherein the isothermal nucleic acidamplification method is selected from Nucleic Acid Sequence BasedAmplification (NASBA), Transcription Mediated Amplification (TMA),Helicase Dependent Amplification (HDA), Recombinase polymeraseamplification (RPA), Strand Displacement Amplification (SDA),Loop-mediated Isothermal Amplification (LAMP), Chimera DisplacementReaction (RDC), Isothermal Chimeric Amplification of Nucleic Acids(ICAN), SMart Amplification Process (SMAP), Linear IsothermalMultimerization Amplification (LIMA), or Self Extending Amplification(SEA).
 43. The method of claim 35, wherein the first nucleic acidamplification is Loop-mediated Isothermal Amplification (LAMP).
 44. Themethod of claim 35, wherein step (a) includes a forward and reverseloop-forming primer.
 45. The method of claim 44, wherein step (a)includes a forward and reverse displacement primer.
 46. The method ofclaim 35, wherein the second isothermal nucleic acid amplificationreaction is LAMP-STEM.
 47. The method of claim 35, wherein the methodcomprises dividing (b) into at least an additional secondary reaction,wherein the additional secondary reaction includes a mismatch secondaryprimer.
 48. The method of claim 45, wherein the mismatch primercomprises a 3′ end nucleotide mismatch or 3′ end nucleotidecomplementary to an allele, mutation or polymorphism different from thesite-specific primer.
 49. The method of claim 35, wherein step (e)comprises detecting and comparing in real-time the amplification ratesof all secondary reactions, wherein an enhanced relative rate ofamplification as compared to the other two reactions indicates thepresence of the site of interest that is complementary to the secondaryprimer.
 50. The method of claim 35, wherein the step of performing thesecond nucleic acid amplification reaction includes amplification in thepresence of a fluorescent label or dye, thereby labeling theamplification products.
 51. The method of claim 50, wherein the step ofdetecting and comparing in real-time the amplification rates includesdetecting and comparing a fluorescent signal from each of the reactions.52. The method of claim 35, wherein a slow-rate isothermalpre-amplification is performed in the first-stage followed by multiple,discrete second-stage amplification and detection reactions performed inparallel directly on the products from the first-stage primary ampliconamplification product.
 53. The method of claim 35, wherein thefirst-stage amplification reaction comprises site-specific primers suchthat a first-stage pre-amplification reaction is selective for aparticular site of interest, and only if that site or region exists inthe sample will rapid amplification of the template proceed at thesecond stage amplification reaction.
 54. The method of claim 35, whereinthe second-stage amplification reaction comprises multiple site-specificprimers.
 55. The method of claim 35, wherein an amount or volume of thefirst-stage reaction is introduced directly into a plurality of secondreaction chambers.
 56. The method of claim 35, further comprising thesteps of providing a first reaction chamber; performing a first-stagepre-amplification reaction in the first reaction chamber; introducing anamount or volume of the first-stage pre-amp reaction directly into aplurality of second reaction chambers, wherein at least one secondreaction chamber comprises a site-specific primer, and at least onesecond reaction chamber comprises a mismatched primer; and performing ineach second reaction chamber a second-stage amplification reaction. 57.The method of claim 35, wherein the first-stage and second-stageamplification reactions are performed sequentially in the same reactioncontainer or chamber.
 58. The method of claim 35, wherein the targetnucleic acid template is nested within a larger amplified region. 59.The method of claim 35, wherein the first-stage amplification reactioncomprises site-specific primers such that the first-stage amplificationreaction is selective for a particular site of interest, and whereinonly if that site or region exists in the sample will rapidamplification of the template proceed at the second-stage amplificationreaction.